KR101987203B1 - Ganglioglioma-induced animal model and drug screening using the same - Google Patents

Abstract

The present invention relates to a biomarker of brain metastasis, a composition for diagnosing brain metastasis, an animal in which brain metastasis is induced, and a composition for preventing or treating brain metastasis. More particularly, the present invention relates to a BRAF mutant protein and a nucleic acid molecule, The present invention relates to a composition for diagnosing cerebral metastasis comprising an agent capable of inhibiting BRAF mutation, a composition capable of inducing brain metastasis transformed with the BRAF mutant nucleic acid molecule, and a composition for preventing or treating brain metastasis comprising an activity inhibitor of a BRAF mutant protein.

Description

Ganglioglioma-induced animal model and drug screening using the same [

The present invention relates to a diagnostic and therapeutic use of a ganglioneoclastoma using a biomarker of a ganglioneoclastoma, and more particularly, to a method for diagnosing and treating a ganglioneoclastoma comprising a BRAF mutant protein and a nucleic acid molecule, an agent capable of detecting the protein or nucleic acid molecule A composition for diagnostic use, an animal derived from the ganglion cell line transformed with the BRAF mutant nucleic acid molecule, and a composition for preventing or treating a ganglioneocarcinoma including an activity inhibitor of a BRAF mutant protein.

Epilepsy A group of chronic neurological diseases in which some neurons develop epileptic seizures repeatedly due to seizure-induced over-electricity in a short period of time, and are accompanied by neurobiological, psychological, cognitive, and social changes Is a serious neurological disorder.

It is the third most common neurological disorder following Alzheimer's and Stroke, with about 0.5% to 2% of the world's population having epilepsy. In addition, there are 45 new cases per 100,000 people worldwide, and about 30 ~ 400,000 cases of epilepsy are estimated to occur in Korea, and about 20,000 cases of new epilepsy occur each year . It is also known that 70% of all epilepsy starts at the age of pediatric adolescents, especially in infancy. The incidence and prevalence are highest in the first year of life, rapidly declining, and suddenly increase in the U-shape in elderly people over 60 years of age. The prevalence of life-long seizures is 10-15%.

Among the epilepsies, epilepsy which does not respond to the antiepileptic drugs developed until now is called intractable epilepsy, accounting for about 40% of total epilepsy.

Malignations of Cortical Developments (MCD), ganglioglioma (GG) and hippocampal sclerosis (HS), or Sturge weber syndrome (SWS) ) Are known.

Ganglioneuccaloma is one of the major causes of intractable epilepsy that does not respond to medication, accounting for approximately 80% of childhood intractable epilepsy. This is the most common nerve cell tumor of the central nervous system and is a benign tumor with dysplasticity in both nerve cell and glia. Most of them occur in pediatric patients, with recurrent episodes of brain metastasis in nearly 80% of these patients. Surgical resection reduces the number of seizures in patients, but in some patients there is a persistent epileptic seizure. The age at onset is low and the location of the tumor is deep, so surgical treatment is sometimes difficult. In particular, the molecular genetic causes of ganglioneoclastomas are not known, so it is difficult to develop new and effective treatments for ganglionic cell tumors.

Although previous studies have speculated that somatic mutations are specific to gangliosomal tumors, there has not yet been a clear causal relationship between these mutations and the onset of ganglionic ganglione cysts. There is no known biologic mechanism involved.

It is an object of the present invention to provide a BRAF mutant protein and a nucleic acid molecule as biomarkers for diagnosing brain metastasis.

A further object of the present invention is a composition for diagnostic of brain metastasis, a diagnostic kit and a diagnostic method comprising an agent capable of detecting a BRAF mutant protein and a nucleic acid molecule.

Another object of the present invention is to provide a composition for screening an animal which has been transformed with a BRAF mutant nucleic acid molecule, and a method for screening a drug for treating epilepsy or ganglionic cell tumor using the animal.

It is a further object of the present invention to provide a composition for preventing or treating brain metastasis and ganglioneoclastoma comprising an activity inhibitor of a BRAF mutant protein.

The inventors of the present invention discovered brain-specific somatic mutations of ganglioneoclastoma in brain tissue of ganglionic cell tumor patients using the whole exon sequence analysis technique, The present inventors have completed the present invention by confirming that when an animal inhibitor of BRAF mutation protein is administered to the animal model, remarkable treatment of brain metastasis is achieved.

In one aspect of the present invention, the present invention provides a mutant protein comprising an amino acid sequence comprising a mutation substituted with glutamic acid at the 600th valine in the amino acid sequence of SEQ ID NO: 1, or

A composition for diagnosing brain metastasis by ganglioneomas or ganglioneocarcinomas comprising an agent capable of detecting a mutant nucleic acid molecule consisting of a nucleotide sequence comprising a mutation in which the 1799th thymine is substituted with adenine in the nucleotide sequence of SEQ ID NO: .

In another aspect, the present invention provides a method for diagnosing brain metastasis, comprising the steps of: obtaining from a sample of a patient a protein consisting of an amino acid sequence comprising a mutation in which the 600th valine in glutamic acid in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamic acid; Or a nucleic acid molecule consisting of a nucleotide sequence comprising a mutation in which the 1799th thymine is substituted with adenine, in the nucleotide sequence of SEQ ID NO: 2.

In another aspect, the present invention provides a method for inducing brain metastasis induced by ganglioneocarcinoma or ganglioneocelloma, comprising a protein consisting of an amino acid sequence comprising a mutation substituted with glutamic acid at position 600 of the amino acid sequence of SEQ ID NO: ≪ / RTI >

In another aspect, the invention relates to a recombinant vector comprising a nucleic acid molecule comprising a mutation in which, in the nucleotide sequence of SEQ ID NO: 2, the 1799th thymine is replaced with adenine. In another aspect, the present invention relates to a cell into which a recombinant vector comprising a nucleic acid molecule comprising a mutation in which the 1799th thymine is substituted with adenine, in the nucleotide sequence of SEQ ID NO: 2.

In another aspect, the present invention relates to a recombinant vector comprising a nucleotide sequence of SEQ ID NO: 2, wherein the recombinant vector comprising a nucleic acid molecule comprising a mutation in which the 1799th thymine is substituted with adenine is introduced into an embryo of an introduced animal or animal will be.

In another aspect, the present invention provides a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 2, wherein the 1799th thymine is transformed with a recombinant vector comprising a nucleic acid molecule comprising a mutation substituted with adenine, Gt; induced < / RTI >

In another aspect, the present invention provides a method for producing a recombinant vector comprising the steps of: preparing, in the nucleotide sequence of SEQ ID NO: 2, a recombinant vector comprising a nucleic acid molecule comprising a mutation in which the 1799th thymine is substituted with adenine;

And transforming the vector into a mouse. The present invention also relates to a method for producing an animal in which glial cells are induced by gangliosoma.

In another aspect, the present invention relates to a method for treating brain injury, comprising administering a candidate agent for treatment of brain metastasis to an animal in which brain injury is induced by the ganglioneoclastoma or ganglioneocarcinoma, The present invention relates to a method for screening a therapeutic agent for brain inflammation by a cell tumor or ganglion cell tumor.

In another embodiment, the present invention provides a method for preventing or treating brain metastasis by ganglioneoclastoma or ganglioneocarcinoma comprising the activity inhibitor of a protein comprising an amino acid substituted with glutamic acid at position 600 of the amino acid sequence of SEQ ID NO: 1 ≪ / RTI >

Hereinafter, the present invention will be described in more detail.

The present invention relates to a diagnostic biomarker for intractable brain metastasis, for example, refractory epilepsy caused by ganglionic ganglion (GG), using a brain lesion-specific somatic mutation of ganglioneoclastoma.

The present invention relates to a biomarker for diagnosing brain metastasis and its use. More specifically, the present invention relates to a BRAF gene in which a mutation of a nucleotide sequence occurs or a BRAF protein in which a mutation of an amino acid sequence occurs due to a mutation of a nucleotide sequence. The present invention also relates to a composition and a kit for diagnosing brain metastasis comprising a preparation capable of detecting the gene or protein. The present invention also relates to a method for detecting the gene and protein as biomarkers for the diagnosis of brain metastasis from a sample of a patient to provide information necessary for diagnosis of brain metastasis.

The term "brain metastasis" in the present invention means a chronic disease in which a part of nerve cells generates excessive electricity in a short period of time and repeated seizures occur. In the present invention, the brain metastasis may include intractable brain metastasis, and the brain metastasis may be refractory brain metastasis due to ganglionic cell (GG).

Based on the analysis of existing cancer data, the inventors of the present invention found that the brain tumor of the lower stage of the pediatric population has a higher proportion of the intractable epilepsy than the adult brain tumor, Analysis of brain tissue samples of ganglioglioma (GG) patients with intractable epilepsy, which had no effect on conventional antiretroviral therapy, revealed that 600 of the BRAF proteins composed of amino acids of SEQ ID NO: 1 (Hereinafter referred to as BRAF V600E) mutant of BRAF V600E substituted with glutamic acid, which is the second amino acid of the BRAF V600E amino acid, and the gene coding the BRAF V600E amino acid and the amino acid was diagnosed as a brain tumor- It can be used as a biomarker panel for

The BRAF V600E mutation was not found in the patient's blood and was found specifically in brain tissue samples. In addition, it was confirmed that the genetic mutation rate in the ganglioneoclastoma patient group was present in a ratio of 30 to 70%, and that the ratio of the mutant gene to the normal allele gene present in each patient was 5% to 35% (Table 3).

Because the tumor used in the experiment was a benign tumor and the number of copies of the mutant cells was small, no mutation was found except for the BRAF V600E mutation as confirmed by full-length exocytosis. In other words, only BRAF V600E single mutation was confirmed to be associated with ganglioneoclastoma, thus confirming that BRAF V600E has a very strong correlation with phenotype in ganglionic cell tumor (Example 2).

The intrathoracic epilepsy of the present invention may be caused by a ganglioneoclastoma. Specifically, the ganglioneoclastoma may be a brain somatic mutation-associated ganglion cell, and preferably the brain somatic mutation may be a brain-specific BRAF V600E mutation, It is not.

The term " brain somatic mutation " in the present invention means that a mutation of a base sequence occurs at one or more positions in a wild type gene. For example, an amino acid variation of a BRAF gene or a protein corresponding to these genes.

The brain metastasis caused by the ganglioneoclastoma or ganglioneoclastoma may not be caused by the activation of the mTOR signaling pathway and the brain metastasis by the ganglioneocyte or ganglion cell tumor may be caused by the mutated protein or the mutated nucleic acid molecule May be caused by expression in nerve cells rather than the glioblastoma, and the brain metastasis by the ganglioneocarcinoma or ganglioneocarcinoma may be caused by expression of the mutant protein or the mutated nucleic acid molecule in the neuron of the embryonic development stage But is not limited thereto.

The embryo development step may refer to a time to the pus, and specifically, a time before the organ is formed.

BRAF gene or protein is known as a protein kinase that plays a role in signaling cell division from the cell membrane to the nucleus. It functions to phosphorylate MEK kinase and ERK kinase and to transmit MAP kinase signal. The amino acid sequence of human BRAF is described in SEQ ID NO: 1, the NCBI accession number in SEQ ID NO: 1 is NP_004324.2, the nucleotide sequence encoding SEQ ID NO: 1 is in SEQ ID NO: 2, The NCBI accession number is NM_004333.4. The amino acid sequence of the human mus musculus BRAF is shown in SEQ ID NO: 3, the NCBI accession number of SEQ ID NO: 3 is NP_647455.3, and the nucleotide sequence encoding SEQ ID NO: 3 is SEQ ID NO: And the NCBI accession number of SEQ ID NO: 2 is NM_139294.5.

In the present invention, a preferred example of a brain somatic mutation means that a mutation has occurred in the amino acid sequence of SEQ ID NO: 1 which is a wild-type human BRAF gene. For example, in the amino acid sequence of SEQ ID NO: 1, it may refer to a protein (BRAF V600E) consisting of an amino acid sequence including a mutation in which the 600th valine is substituted with glutamic acid. In another embodiment, the brain somatic mutation in the present invention is a nucleic acid molecule consisting of a nucleotide sequence comprising a mutation in which the 1799th thymine is substituted with adenine in the nucleotide sequence of SEQ ID NO: 2, which is a nucleotide sequence encoding a wild type BRAF protein have.

The mutation of mouse BRAF corresponding to human BRAF may mean a protein (BRAF V600E) consisting of an amino acid sequence comprising a mutation in which the 637th valine is substituted with glutamic acid in the amino acid sequence of SEQ ID NO: 3, The nucleotide sequence of SEQ ID NO: 4, which is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 4, and the nucleotide sequence of the nucleotide sequence including the mutation of the 1910th thymine with adenine.

In one embodiment of the present invention, in order to produce a mouse expressing a mutation of BRAF, a protein (BRAF V600E) consisting of an amino acid sequence comprising a mutation substituted with glutamic acid at position 637 of the amino acid sequence of SEQ ID NO: 3, In the nucleotide sequence of SEQ ID NO: 4, which is the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3, a nucleic acid molecule consisting of the nucleotide sequence including the mutation of the 1910th thymine with adenine was used.

However, since mouse BRAF V637E is commonly referred to as BRAF V600E as human BRAF V600E, BRAF V600E is described in this specification for a mouse expressing the BRAF V637E mutation.

In addition, the mutant protein may include additional mutations within a range that does not totally alter the activity of the molecule. Amino acid exchanges in proteins and peptides that do not globally alter the activity of the molecule are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). In some cases, the BRAF mutant protein may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like.

The present invention provides a mutant protein comprising an amino acid sequence comprising a mutation substituted with glutamic acid at the 600th valine in the amino acid sequence of SEQ ID NO: 1,

A composition for diagnosing brain metastasis by ganglioneomas or ganglioneocarcinomas comprising an agent capable of detecting a mutant nucleic acid molecule consisting of a nucleotide sequence comprising a mutation in which the 1799th thymine is substituted with adenine in the nucleotide sequence of SEQ ID NO: .

As used herein, the term "diagnosis " means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to identify the onset or the likelihood of developing epryptic brain metastasis by ganglionic or ganglionic cell tumors.

In one embodiment of the present invention, in order to accurately reflect the model of tumor-derived epilepsy in an animal model, ganglion cell types known to be the most likely cause of epilepsy-related tumors in children are selected, And a blood sample were obtained, and the BRAF V600E mutation was specifically confirmed (Examples 1 and 2) in the brain tissue sample, so that the BRAF V600E mutation can be used as a diagnostic marker for brain metastasis, Or as a diagnostic marker for glioblastoma caused by ganglioneoclastoma.

In the present invention, "agent capable of detecting a gene" means a substance that can be used to detect a BRAF mutation gene in a sample of a patient. For example, in the nucleotide sequence of SEQ ID NO: 2, a primer, a probe, an antisense oligonucleotide, or the like complementary to a gene consisting of a nucleotide sequence including a mutation in which the 1799th thymine is substituted with adenine . In the nucleotide sequence of SEQ ID NO: 2, the primer, probe, or antisense nucleic acid specifically binds to the nucleotide sequence including the mutation in which the 1799th thymine is substituted with adenine, and does not specifically bind to the nucleotide sequence of the other nucleic acid material desirable.

Here, complementary binding means that the antisense nucleic acid is sufficiently complementary to selectively hybridize to the BRAF mutant gene target under a predetermined hybridization or annealing condition, preferably physiological conditions, and is substantially complementary the term " substantially complementary " and " perfectly complementary "

For example, the agent used to detect BRAF variant gene biomarkers herein may be an antisense nucleic acid.

The term "antisense nucleic acid" means a nucleic acid-based molecule having a complementary sequence to a target BRAF mutant gene and capable of forming a dimer with the BRAF mutant gene, and is used for detecting the BRAF mutant gene biomarker Can be used.

Alternatively, the agent used to detect the BRAF mutant gene biomarker of the present invention is a primer pair or a probe, and since the nucleotide sequence of the BRAF mutant gene is known in the present specification, A primer or a probe that specifically amplifies a specific region of these genes can be designed.

The term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming base pairs with a complementary template and functioning as a starting point for template strand copying. Lt; RTI ID = 0.0 > 50 < / RTI > Primers are usually synthesized but can also be used in naturally occurring nucleic acids. The sequence of the primer does not necessarily have to be exactly the same as the sequence of the template, but is sufficiently complementary that it can hybridize with the template. Preferably, the primer of the present invention can be a primer capable of amplifying a BRAF mutant gene.

In another example, the agent used to detect the BRAF variant gene biomarkers herein may be a probe. The term "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a short period of a few nucleotides or a few hundred nucleotides that can specifically bind to mRNA, and is labeled to confirm the presence or absence of a specific mRNA . The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe.

In the present invention, hybridization can be performed using a probe complementary to the BRAF genetic mutation, and diagnosis can be made by hybridization. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.

In the present invention, "an agent capable of detecting a protein" means a substance that can be used to detect a BRAF mutant protein in a sample of a patient. Preferably, the BRAF variant protein may be a specific compound or synthetic target. As a specific example, in the amino acid sequence of SEQ ID NO: 1, the 600th valine may be an antibody or an aptamer specific for a protein consisting of an amino acid sequence comprising a mutation substituted with glutamic acid. Preferably, the antibody may be a monoclonal antibody or a polyclonal antibody.

The term "antibody ", as it is known in the art, refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a BRAF mutant protein, which is a marker of the present invention. Such an antibody may be obtained by cloning a BRAF mutant gene into an expression vector according to a conventional method, A BRAF mutant protein encoded by the BRAF mutant gene can be obtained and can be prepared from the obtained BRAF mutant protein by a conventional method. Also included are partial peptides that can be made from the BRAF mutant proteins, and the partial peptides of the invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 or more amino acids. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, a monoclonal antibody or a part thereof having antigen binding ability is included in the antibody of the present invention and includes all immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

Antibodies used in the detection of brain metastasis diagnostic biomarkers of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains do. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In addition, the present invention can be implemented in the form of a kit in the form of a composition for diagnosing brain metastasis, which comprises a preparation capable of detecting a BRAF mutant gene or a BRAF mutant protein.

The kit of the present invention can detect BRAF mutation gene or BRAF mutant protein which is a biomarker for diagnosing brain metastasis. The kit of the present invention may further comprise one or more other component compositions suitable for the assay method as well as antibodies recognizing BRAF mutant genes or BRAF mutant proteins, primers, probes, antisense nucleic acids or optionally BRAF variant protein- Device may be included.

As a specific example, the kit for detecting a BRAF mutant gene in the present invention may be a kit for the diagnosis of brain metastasis, which includes essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, a reagent for preparing a fluorescent-labeled probe, a preparation, an enzyme, and the like. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof. In addition, the kit for detecting the BRAF mutation gene may be a kit containing essential elements necessary for performing the PCR. The PCR kit contains, in addition to each primer pair specific for the BRAF variant gene, a test tube or other appropriate container, reaction buffer (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq polymerase Such as enzymes, DNase, RNAse inhibitors, DEPC-water, sterile water, and the like. It may also contain a primer pair specific to the gene used as a quantitative control.

As another specific example, in the present invention, a kit for detecting a BRAF mutant protein may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, etc. for immunological detection of the antibody have. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, a slide glass made of glass, etc. The coloring enzyme may be peroxidase, Alkaline Phosphatase may be used as the fluorescent substance, FITC, RITC or the like may be used as the fluorescent substance, and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ) Or OPD (o-phenylenediamine), TMB (tetramethylbenzidine) can be used.

In order to provide information necessary for the diagnosis of brain metastasis, the present invention provides a method for diagnosing brain metastasis, which comprises extracting a protein consisting of an amino acid sequence comprising a mutation substituted with glutamic acid at position 600 with a 600th valine in the amino acid sequence of SEQ ID NO: 1 or a protein comprising a nucleotide sequence of SEQ ID NO: , A method for detecting a nucleic acid molecule comprising a nucleotide sequence comprising a mutation in which the 1799th thymine is substituted with adenine.

The method for detecting the nucleic acid molecule may include amplifying a nucleic acid from a sample of a patient, and determining a base sequence of the amplified nucleic acid.

The sample may be a patient's brain tissue sample, but is not limited thereto.

In one aspect of the present invention, there is provided a method for detecting a BRAF mutant gene or a BRAF mutant protein from a sample of a patient to provide information necessary for diagnosis of brain metastasis.

More specifically, it can be performed by a method of detecting a BRAF mutant gene or a BRAF mutant protein, and separation of genomic DNA or total protein from a patient sample can be performed using a known process .

The term "patient sample" in the present invention includes tissues such as a BRAF mutant gene or a BRAF mutant protein, cells, and the like. Preferably, it may be, but is not limited to, brain tissue.

Preferably, a method for detecting a BRAF mutant gene from a sample of a patient can be performed by a method comprising amplifying a nucleic acid from a sample of a patient, and determining a base sequence of the amplified nucleic acid.

Specifically, the step of amplifying the nucleic acid may be performed using a polymerase chain reaction (PCR), a multiplex PCR, a touchdown PCR, a hot start PCR, a nested PCR, a booster PCR , Real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), inverse polymerase chain reaction, vectorette ) PCR, thermal asymmetric interlaced PCR (TAIL-PCR), ligase chain reaction, repair chain reaction, transcription-mediated amplification, self-retaining base sequence replication or selective amplification of the target base sequence .

In addition, the step of determining the nucleotide sequence of the amplified nucleic acid may be performed by a method selected from the group consisting of Sanger sequencing, Maxam-Gilbert sequencing, Shotgun sequencing, pyrosequencing, hybridization by microarray, PCR can be performed by allele-specific PCR, dynamic allele-specific hybridization (DASH), PCR extension analysis, TaqMan technique, automated sequencing, or next generation sequencing. The next generation nucleotide sequence analysis can be performed using a nucleotide sequence analysis system widely used in the art. For example, 454 GS FLX from Roche, Genome Analyzer from Illumina, and SOLID Platform from Applied Biosystems can be used.

As another example, a method for detecting a BRAF mutant protein from a patient sample includes Western blotting using an antibody specifically detecting the amino acid mutation, ELISA, radioimmunoassay, radioimmunoprecipitation, Ouchteroni immunodiffusion, But are not limited to, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS, protein chips, and the like. Through the above analysis methods, it is possible to identify an antigen-antibody complex between the BRAF mutant protein and an antibody thereof, and to diagnose an EA-antibody complex between the BRAF mutant protein and the antibody.

The term " antigen-antibody complex "as used herein means a conjugate of a BRAF mutant protein and an antibody specific thereto, and the formation of an antigen-antibody complex can be measured through a signal of a detection label. Such detection labels may be selected from the group consisting of enzymes, minerals, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto.

In one embodiment, the antigen-antibody complex between the BRAF mutant protein and the antibody is measured using ELISA. Further, preferably, one or more antibodies against BRAF mutant proteins are arrayed at predetermined positions on the substrate to use protein chips immobilized at high density. Methods for analyzing a sample using a protein chip include a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein to confirm the presence of the protein, Can be confirmed.

Preferably, the method of detecting a BRAF mutant protein from a sample of a patient comprises: separating the whole protein from a sample of the patient; Analyzing the amino acid sequence of the separated protein and comparing it with a reference sequence. Also preferably, it can be performed by a method for measuring the activation of BRAF protein increased by BRAF genetic variation. Through the above detection methods, when a BRAF mutation gene or a BRAF mutant protein is detected, it can be diagnosed by brain metastasis.

The present invention relates to a BRAF V600E recombinant vector comprising a nucleic acid molecule consisting of a nucleotide sequence comprising a mutation in which the 1799th thymine is substituted with adenine, in the nucleotide sequence of SEQ ID NO: 2.

In one embodiment of the present invention, a gene in which the 1910th thymine is substituted with adenine in the nucleotide sequence of SEQ ID NO: 4 was amplified using a known site-directed mutagenesis method and ligated to Cre recombinase-dependent loxP sequence, A conditional mutant transgenic mouse expressing the Cre-dependent BRAF V600E protein was prepared by homologous recombination in the embryonic cells. When the mice obtained by crossing the prepared mouse and the tdTomato mouse reached the 14th day of pregnancy (E14) Mice that were exposed to the uterine horn and injected Cre recombinase into the lateral ventricle of each embryo to induce BRAF V600E mutation by Cre recombinase Respectively.

The mouse expresses BRAF V637E protein in which the 637 valine is replaced with glutamic acid in the amino acid sequence of SEQ ID NO: 3, or the mouse expresses the gene in which the 1910th thymine is substituted with adenine in the nucleotide sequence of SEQ ID NO: 4 have.

However, since the mouse BRAF V637E is commonly referred to as BRAF V600E in the same manner as the human BRAF V600E, the mouse BRAF V637E is described as BRAF V600E in the present invention.

The recombinant vector may be a vector which can be expressed in a mammal or rodent.

The present invention relates to a composition for inducing brain metastasis by ganglioneocarcinoma or ganglioneocarcinoma, which comprises a protein consisting of an amino acid sequence comprising a mutation substituted with glutamic acid at position 600 in the amino acid sequence of SEQ ID NO: 1.

The composition for inducing brain metastasis by the ganglioneoclastoma or ganglioneoclastoma is a mutant protein specifically found in brain metastasis caused by ganglioneocarcinoma or ganglioneoclastoma, wherein the 600th valine in the amino acid sequence of SEQ ID NO: 1 is glutamic acid And a protein consisting of an amino acid sequence including a substituted mutation. The protein consisting of the amino acid sequence comprising a mutation in which the 600th valine is replaced with glutamic acid in the amino acid sequence of SEQ ID NO: 1 is the nucleotide sequence of SEQ ID NO: 2, the nucleotide sequence comprising the mutation in which the 1799th thymine is substituted with adenine It can be coded.

As used herein, the term "induction " means inducing a change from a normal state to a pathological state. For the purpose of the present invention, induction is a transition from a state in which no epilepsy occurs to a state in which epilepsy occurs. Specifically, in the amino acid sequence of SEQ ID NO: 1, a composition comprising a protein consisting of an amino acid sequence including a mutation in which the 600th valine is substituted with glutamic acid may be injected to induce brain metastasis. In addition, in the nucleotide sequence of SEQ ID NO: 2, a composition comprising a gene consisting of a nucleotide sequence including a mutation in which the 1799th thymine is substituted with adenine may be injected to induce brain metastasis. The mutated protein of SEQ ID NO: 1 or the gene of SEQ ID NO: 2 in which the gene sequence is mutated may be injected to induce brain metastasis.

The brain metastasis caused by the ganglioneoclastoma or ganglioneoclastoma may not be caused by the activation of the mTOR signaling pathway and the brain metastasis by the ganglioneocyte or ganglion cell tumor may be caused by the mutated protein or the mutated nucleic acid molecule May be caused by expression in nerve cells rather than the glioblastoma, and the brain metastasis by the ganglioneocarcinoma or ganglioneocarcinoma may be caused by expression of the mutant protein or the mutated nucleic acid molecule in the neuron of the embryonic development stage But is not limited thereto.

The embryo development step may refer to a time to the pus, and specifically, a time before the organ is formed.

In one embodiment of the present invention, the brain metastasis caused by the ganglioneoclastoma or ganglioneoclastoma is independent of the BRAF V600E mutation originated from the glioblastoma, and BRAF V600E mutation in the neuron plays an important role in the epileptogenesis mechanism And BRCA V600E mutations were observed only in the diploid cells, it was confirmed that the cell proliferative capacity and benign tumors were increased. The above results indicate that BRAF V600E mutation in the glioma is caused by BRAF V600E mutation occurring in the neuron of the ganglion cell line or ganglion cell line of the present invention, It was found that it induces cell proliferation rather than inducing brain metastasis by glial cell type (Example 6).

In one embodiment of the present invention, the brain metastasis caused by the ganglioneoclastoma or ganglioneoclastoma is induced not by the BRAF V600E mutation expressed in adulthood, but by the BRAF V600E mutation expressed in the embryo development stage before the tracheal development (Example 5).

In addition, in one embodiment of the present invention, it was confirmed that the brain metastasis by the ganglioneoclastoma or ganglioneocarcinoma was not caused by the mTOR signaling pathway (Example 9).

In the nucleotide sequence of SEQ ID NO: 2, the present invention may be a recombinant vector comprising a nucleic acid molecule comprising a mutation in which the 1799th thymine is substituted with adenine.

The present invention relates to a nucleotide sequence of SEQ ID NO: 2 in which a recombinant vector containing a nucleic acid molecule comprising a mutation in which the 1799th thymine is substituted with adenine is introduced. The cell may be a brain cell.

The present invention relates to an embryo in which a recombinant vector comprising a nucleic acid molecule comprising a mutation in which the 1799th thymine is substituted with adenine is introduced in the nucleotide sequence of SEQ ID NO:

The embryo may be a mammal or a rodent, excluding humans, and the embryo may be an embryo at the stage of development or development of the brain.

The present invention relates to a method for producing a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 2, wherein the 1799th thymine is transformed with a recombinant vector comprising a nucleic acid molecule comprising a mutation substituted with adenine to induce brain metastasis by ganglioneoclastoma or ganglionic cell tumor , And animals other than humans.

The present invention relates to a transgenic animal with said recombinant vector.

In the present invention, the term "transgenic animal" means an animal in which intracellular BRAF V600E protein activity is increased relative to that of normal cells, and a vector expressing a BRAF protein having a mutated amino acid sequence Lt; RTI ID = 0.0 > intracellular < / RTI > The transgenic animal in which the brain metastasis has occurred can be effectively used as an animal model for brain tumors.

The animal may be a mammal or a rodent other than a human, and may be one in which brain metastasis is induced by a ganglioneoclastoma or ganglioneocarcinoma.

The present invention provides a method for producing a recombinant vector comprising the steps of: preparing, in the nucleotide sequence of SEQ ID NO: 2, a recombinant vector comprising a nucleic acid molecule comprising a mutation in which the 1799th thymine is substituted with adenine;

And transforming the vector into a mouse. The present invention also relates to a method for producing an animal in which glial cells are induced by gangliosoma.

The method of introducing the recombinant vector is not particularly limited. For example, the vector may be inserted into a cell through a method such as transformation, transfection or transduction. The vector inserted into the cell can produce a BRAF protein in which the amino acid sequence is mutated due to constant gene expression in the cell.

The recombinant vector may be introduced into the brain of the embryo during the period during which the cortical layer is formed in the embryo, but the present invention is not limited thereto.

In one specific embodiment of the present invention, a conditionally mutant transgenic mouse capable of expressing a gene in which the 1910th thymine is adenine-dependent is introduced in the nucleotide sequence of SEQ ID NO: 4, and a plasmid having the Cre recombinase Mice were transfected into the uterus to prepare mouse animal models in which brain tumors were induced.

The present invention relates to an animal which has been transformed with a recombinant vector containing a nucleotide sequence encoding an amino acid substituted with glutamic acid at position 600 of the amino acid sequence of SEQ ID NO: 1. The animal may be a mammal or a rodent, other than a human.

The term " animal model "or" disease model "in the present invention refers to a disease model having a specific disease similar to a human disease so as to identify a pathogen, Means an animal. Animal for use as an animal model can predict the same effects as in humans, can easily be made, and is reproducible. It should also proceed in a manner similar to or similar to the etiology of human disease. Therefore, animals that are similar to mammalian vertebrates such as humans, similar to organs such as organs, immune system, body temperature, etc., and suffer from diseases such as hypertension, cancer, immunodeficiency and the like are suitable as animal models. Such an animal is preferably a mammal such as a horse, a sheep, a pig, a goat, a camel, a nutrition, a dog, a rabbit, a mouse, a rat, a guinea pig, a hamster and the like, more preferably a rodent such as a mouse, a rat, a guinea pig,

In one embodiment of the present invention, BRAF V600E has a very strong correlation with the phenotype of ganglioneoclastoma patients. Based on this finding, when the normal BRAF gene is replaced with BRAF V600E mutation in animal models, (Example 4).

In one embodiment of the present invention, prior to confirming that a phenotypic phenotype is present in the BRAF gene replacement model, the patient tissue is stained with a marker specific to a neuron cell and a cell specific marker, By confirming the presence or absence of the BRAF V600E mutation by the method of dissecting with a microscope, it was confirmed that the BRAF V600E mutation originates from both neuronal cells and cell lineages (FIGS. 2A and 2B). From the above results, it can be deduced that the BRAF V600E mutation occurred in a common ancestor of both neurons and glia (Example 3).

In one embodiment of the present invention, in the mouse animal model prepared above. Electroencephalography (video-EEG) surveillance was performed 3 weeks after the embryo was enucleated by electroporation of the plasmid with Cre recombinase in the lateral ventricle of the embryonic mouse on day 14 (E14). As a result, Spontaneous seizures accompanied by epileptic seizures were observed in mice injected with the plasmid in which the mutation gene with the base sequence mutation of the invention was inserted (FIGS. 3B and 3C). In addition, the brain of the animal model mouse was cut, and the tissue activity was measured using a multi-channel electrode analyzer. As a result, unlike the control group, the BRAF V600E mutant brain tissue was characterized by spontaneous activity waves, And a synchronized burst firing (FIG. 3C) in which a short cycle of high amplitude energy is emitted appears (Example 4).

In one embodiment of the present invention, the GFP-positive cells of the cerebral region induced by electroporation of the BRAF V600E mutation are accompanied by neuronal dysplasias characteristic of glioblastoma cells, 5a), the shape of the cells is distorted (Fig. 5b), and the arrangement of the cell shapes and branches of the nerve cells is arranged in a random direction unlike the normal neurons (Fig. 5c).

In addition, in one embodiment of the present invention, a plasmid gene having a Cre recombinase gene was crossed with a conditional floxed tdTomato mouse in order to track daughter cells of a BRAF V600E mutation-bearing neural stem cell, 6a). As a result of the follow-up of the daughter cells, it was confirmed that the astrocytes-related astrocytes or glial cells were significantly increased (Fig. 6b) in the neuronal cells with the BRAF V600E mutation.

From the above results, it can be seen that the mouse produced by the method of the present invention is suitable as an animal model in which ganglionic cell disease is induced, since neuronal dendrites and increased number of glioblast cells are observed.

The animal model of EEG of the present invention can be effectively used for research on gene function, molecular mechanism of EEG, and search for new anticonvulsant drug.

The present invention relates to a method of screening for a therapeutic agent for brain inflammation comprising the step of administering a candidate agent for treatment of cerebral apoplexy to an animal in which brain injury is induced by the ganglioneoclastoma or ganglioneoclastoma, and then confirming whether cerebral angiopathy is relieved.

A substance that indirectly or directly alleviates the episodes of cerebral apoplexy after administration of the candidate substance for treatment of cerebral apoplexy to the animal in which the cerebral trauma has been induced can be selected as an agent for treatment of cerebral infarction. In other words, after measuring the symptoms of epilepsy in the absence of candidates for treating epilepsy, comparing epilepsy symptoms in the presence of candidates for treatment of epilepsy, and comparing epileptic symptoms with epileptic symptoms, In the absence of candidate substances for treatment of hyperglycemia, a substance that alleviates symptoms can be predicted as an agent for treating epilepsy.

The present invention relates to a composition for preventing or treating brain metastasis caused by a ganglioneocyte or ganglion cell tumor comprising an activity inhibitor of a protein comprising an amino acid substituted with glutamic acid at position 600 of the amino acid sequence of SEQ ID NO: 1.

Specific examples of the activity inhibitor of a protein comprising an amino acid substituted with glutamic acid at position 600 of the amino acid sequence of SEQ ID NO: 1 according to the present invention include one selected from the group consisting of Vemurafenib or a salt thereof and Dabrafenib or a salt thereof Or more.

The inhibitor may be Vemurafenib or Dabrafenib, and the Vemurafenib may also be referred to as PLX4032, PLX4720, or Zelboraf.

In one embodiment of the present invention, it was confirmed that when BRAF V600E mutation was introduced into cells, BRAF protein was overactivated, resulting in intractable brain metastasis. When the activity inhibitor of BRAF V600E protein was administered, (Example 8) The prevention, amelioration, or treatment of intractable epilepsy caused by intractable epilepsy, and the prevention, improvement, or therapeutic effect of ganglioglioma (GG) .

The present invention provides a composition, kit, or method for preventing, ameliorating or treating intractable epilepsy and for preventing, ameliorating or treating ganglioglioma (GG), which is a causative disease of these intractable epilepsy. Preferably, said intrasorguler lineage episodes are for prevention, treatment and / or amelioration of cerebral somatic mutation-related intractable epilepsy.

The brain metastasis caused by the ganglioneoclastoma or ganglioneoclastoma may not be caused by the activation of the mTOR signaling pathway and the brain metastasis by the ganglioneocyte or ganglion cell tumor may be caused by the mutated protein or the mutated nucleic acid molecule May be caused by expression in nerve cells rather than the glioblastoma, and the brain metastasis by the ganglioneocarcinoma or ganglioneocarcinoma may be caused by expression of the mutant protein or the mutated nucleic acid molecule in the neuron of the embryonic development stage But is not limited thereto.

The embryo development step may refer to a time to the pus, and specifically, a time before the organ is formed.

The composition comprising the inhibitor may be characterized in that seizures caused by ganglionic neuroblastoma or ganglioneocarcinoma are reduced.

In the present invention, the inhibitor may be Vemurafenib, or Dabrafenib, and the inhibitor may include both a derivative or analogue of the compound and a pharmaceutically acceptable salt or hydrate thereof.

The pharmaceutically acceptable salt or hydrate may be a salt or hydrate derived from an inorganic or organic acid. Examples of the salt include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, glycolic acid, lactic acid, pyruvic acid, But are not limited to, acetic acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, citric acid, tartaric acid, P-toluenesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and toluenesulfonic acid. This hydrate may mean that Vemurafenib is formed in association with water molecules.

In the present invention, "treatment" is meant to include alleviating or ameliorating symptoms, reducing the extent of disease, delaying or alleviating disease progression, improving, alleviating or stabilizing disease states, partial or complete recovery, Can be used to mean both inclusive.

Symptoms associated with brain metastasis by the ganglioneoclastoma or ganglioneoclastoma are those in which neurons fail to migrate to the proper brain region during development of the brain, which is characterized by spontaneous seizures, behavioral seizures, Abnormal neuronal cell development, and the like.

Therefore, the treatment according to the present invention can be applied to a patient suffering from brain gonadal hyperplasia or ganglioneocarcinoma by administering BRAF V600E protein activity inhibitor such as Vemurafenib, for example, to show the number of spontaneous seizures, behavioral seizures or EEG seizures , And to reduce abnormal neuronal activity or abnormal signals in the cerebrum.

Depending on the use of the pharmaceutical composition of the present invention and the method of use, the effective amount of the BRAF protein activity inhibitor may be suitably adjusted according to the choice of those skilled in the art.

For example, the pharmaceutical composition may contain the BRAF protein activity inhibitor in an amount of 0.1 to 10% by weight, more preferably 0.5 to 5% by weight, based on the total weight of the total composition.

The BRAF protein activity inhibitor may be included alone in the pharmaceutical composition, or may further include other pharmacologically acceptable additives. The pharmaceutically acceptable excipients which are conventionally used in the preparation include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and also as a pharmaceutically acceptable excipient Include, but are not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives and the like. That is, the pharmaceutically acceptable additives that can be added to the pharmaceutical composition of the present invention may be selected without difficulty by a person skilled in the art depending on the purpose of use, and the amount thereof to be added is within the range that does not impair the purpose and effect of the present invention ≪ / RTI >

The preferred dosage for the patient of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by a person skilled in the art. However, for the desired effect, the extract of the present invention is administered at a dose of 1 mg / kg to 1000 mg / kg, preferably 50 mg / kg to 500 mg / kg, more preferably 150 mg / kg to 300 mg / kg per day Lt; / RTI > The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, or intracerebroventricular injection.

In another aspect, the present invention relates to a food composition for preventing or ameliorating brain metastasis caused by a ganglioneocarcinoma or ganglioneocarcinoma, which comprises a BRAF protein activity inhibitor, for example, Vemurafenib or a salt thereof.

The food composition may be used together with components of other conventional food compositions, and may be suitably used according to conventional methods. The BRAF protein activity inhibitor may be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, when the food composition is prepared, it may be added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight, based on the raw material of the active ingredient. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The food composition may be contained in a health food for the purpose of preventing or ameliorating brain metastasis caused by ganglioneoclastoma or ganglioneocarcinoma, and there is no particular limitation on its kind. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, and may include all health foods in a conventional sense. In addition to the above, the food composition of the present invention may further comprise a pharmacologically acceptable additive. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention relates to a biomarker of brain metastasis, a composition for diagnosing brain metastasis, an animal in which brain metastasis is induced, and a composition for preventing or treating brain metastasis. More particularly, the present invention relates to a BRAF mutant protein and a nucleic acid molecule, The present invention also relates to a composition for preventing or treating brain metastasis comprising an agent for inducing brain metastasis transformed with said BRAF mutant nucleic acid molecule and an activity inhibitor of BRAF mutant protein, The composition for prevention or treatment of brain metastasis comprising the activity inhibitor of the BRAF mutant protein of the present invention can be used to prevent or treat ganglioneocarcinoma or brain metastasis thereof.

FIG. 1A shows that BRAF V600E is significantly present in low-grade brain tumors of children with statistical significance.
FIG. 1B is a diagram showing the MR findings (left) of the glioblastoma cell tumor patient (GG221) and the histopathological findings (right side) of the corresponding lesion in the arrow during surgery.
FIG. 1c is a picture showing preoperative magnetic resonance imaging findings of glioblastoma (GG57, GG163, GG221, GG231, GG249, and GG381).
FIG. 2A is a graphical representation of an experiment on the presence or absence of a BRAF V600E mutation in neurons and glia using a laser-captured microscope dissection method.
FIG. 2B shows the presence or absence of BRAF V600E mutation in neurons (left) and right (right) cells using a laser-captured microscope dissection method.
FIG. 3A is a graph showing that a mouse produced according to an embodiment of the present invention expresses a BRAF V600E mutant gene. FIG.
FIG. 3B is a diagram showing that a spontaneous seizure accompanied by an epileptic seizure occurred in a mouse injected with a plasmid in which a BRAF V600E mutation gene was inserted according to an embodiment of the present invention.
Figures 3c, d, and e illustrate the pathological characteristics characteristic of brain metastasis in brain tissue obtained from mice expressing the BRAF V600E mutant protein produced according to one embodiment of the present invention.
FIG. 4 is a graph showing the results of synchronized burst firing in which mice expressing the BRAF V600E mutant protein produced according to the present invention exhibit spontaneous activity waves and simultaneously emit short-period high amplitude energy in several channels simultaneously to be.
FIG. 5 is a graph showing that neuronal dysplasia, which is characteristic in ganglioneoclastoma, is accompanied in a mouse expressing the BRAF V600E mutant protein produced according to an embodiment of the present invention.
FIG. 6A shows the process of crossing conditional floxed tdTomato mice to track daughter cells of BRAF V600E mutant neural stem cells.
FIG. 6B is a graph showing that the number of glial cell-associated astrocytes or glial cells in BRAF V600E mutant neuronal cells produced according to an embodiment of the present invention was significantly increased compared to that of normal brain tissue.
FIG. 6C is an enlarged photograph of the tissue photograph of FIG. 6B taken at a high magnification.
FIG. 6d shows that CD34 expression was increased by immunohistochemical staining in actual patient tissues.
FIG. 7 is a graph showing that the expression level of CD34 is increased in a mouse expressing the BRAF V600E mutant protein prepared according to an embodiment of the present invention.
FIG. 8 shows immunofluorescence staining (left) and immunohistochemical staining (right) showing increase of CD34 marker in mice expressing the BRAF V600E mutant protein prepared according to an embodiment of the present invention.
FIG. 9 is a diagram showing abnormal lamination of the cortex in a mouse expressing the BRAF V600E mutant protein produced according to an embodiment of the present invention. FIG.
FIG. 10 is a graph showing that a brain model having a BRAF V600E mutation is different from a normal brain tissue when a mouse model is manufactured by the method of the embodiment of the present invention. In the figure at the bottom of Fig. 10, these observations were enlarged, and it was confirmed that there was no change in the amount of whole neurons.
FIG. 11 is a graph showing that the number of cells of the epidermis is increased in the mouse expressing the BRAF V600E mutant only in the epidermal cells according to one embodiment of the present invention.
FIG. 12 shows a method for producing a BRAF V600E mutant mouse inducible by using tamoxifen in adult mice.
Fig. 13 is a diagram showing a method of producing a BRAF V600E mutant mouse inducible using viruses in adult mice.
Fig. 14 is a diagram showing cytologic abnormalities observed when BRAF V600E mutation was induced in adult mice. Fig.
FIG. 15 is a graph showing the decrease in seizure through chronic intracerebroventricular injection (cICV) of a BRAF V600E-specific inhibitor in a mouse expressing a BRAF V600E mutant protein prepared according to an embodiment of the present invention, It is a figure confirmed by measurement of seizure spikes. POD (post operation day) means post-operative day, and ictal seizure means epileptic seizure.
FIG. 16 is a graph showing the results of interictal spikes through video monitoring EEG analysis by continuous intraventricular injection of BRAF V600E-specific inhibitor in mice expressing the BRAF V600E mutant protein prepared according to an embodiment of the present invention. And electrophysiological seizure (electrophysiologic seizure).
FIG. 17 is a graph showing the effect of BRAF V600E-specific inhibitor aICV (acute intracerebroventricular injection) and oral administration (PO, per oral) on mice expressing the BRAF V600E mutant protein prepared according to an embodiment of the present invention In the monitoring EEG analysis, it was confirmed that there was no decrease in seizure. P0
18 is a graph showing the activation of the mTOR signaling pathway in a mouse expressing the BRAF V600E mutant protein prepared according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1. Profiling of dielectric range through analysis of common data

It is known that low grade glioma of the pediatric gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic malignant gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecology gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic gynecologic In order to find out why epilepsy is occurring at a high frequency in pediatric low grade glioma, genome common data such as TCGA (Cancer Genome Atlas) and PeCan (Pediatric Cancer Genomic Data Portal) Mutant genotypes were classified in brain tumors of children.

As a result, as shown in Fig. 1, mutations of genes such as IDH1 and TP53 were remarkably present in adult brain tumors, but BRAF V600E mutations were statistically significant in childhood low-grade brain tumors.

Adult brain tumor Pediatric brain tumor Pediatric neoplasm Number of patients (n) 283 274 73 Gene type Number of mutations (percentage%) IDH1 215 (75.97) 3 (1.09) 1 (1.37) TP53 128 (45.23) 38 (13.87) 0 (0) H3F3A 0 (0) 47 (17.15) 3 (4.11) CIC 26 (9.19) 1 (0.36) 1 (1.37) BRAF 2 (0.71) 5 (1.82) 19 (26.03) ATRX 16 (5.65) 3 (1.09) 1 (1.37) PIK3CA 2 (0.71) 16 (5.84) 0 (0) SMARCA4 8 (2.83) 9 (3.28) 0 (0) ACVR1 0 (0) 16 (5.84) 0 (0) NOTCH1 14 (4.95) 2 (0.73) 0 (0) all 411 140 25

Example 2. BRAF V600E Mutation Confirmation Using Whole Exome Sequencing

2-1. In all five patients Exome  By sequencing BRAF V600E  Identify mutation candidates

In order to accurately reflect the tumor-derived epilepsy patient model in animal models, a ganglion cell tumor known to be the most likely cause of epilepsy-related tumors in children was selected to screen for gene mutations present in the ganglionic cell tumor, Brain tissue and blood samples were taken after surgery of 5 patients with cell tumor (designated GG29, GG30, GG221, GG231, and GG249).

We performed whole-body exome sequencing (read depth 630-672) in brain tissue samples of ganglioneoclastoma patients and selected BRAF V600E, a candidate mutation found in both Strelka and Mutect algorithms of total exome sequencing .

Specifically, a sequencing library was prepared using the Agilent library preparation protocols (Agilent Human All Exon 50 Mb kit) provided by the manufacturer for whole exome sequencing. The prepared sequencing library was sequenced using Hiseq 2000 (Illumina) and sequencing was performed at a depth of 5 times the normal sequencing depth (~ 500x) in order to improve the accuracy of the analysis.

The data after the sequencing was converted into a bam file that can be analyzed using Broad Institute best practice pipleline (https://www.broadinstitute.org/gatk/).

Sample Overall yield (bp) The entire depth of the target region X
The average depth of the target region (X) GG221-Blood 44,917,301,962 630.4 293.35 GG221-Brain 39,837,168,006 559.1 296.33 GG29-Blood 47,943,121,572 579.8 327.14 GG29-Brain 43,394,449,010 596.3 328.11 GG30-Blood 43,443,678,228 609.0 329.51 GG30-Brain 41,317,104,442 609.7 341.22 GG231-Blood 45,607,318,610 630.7 351.96 GG231-Brain 42,488,301,048 640.0 352.19 GG249-Blood 44,944,663,064 669.9 360.64 GG249-Brain 47,736,544,858 672.8 361.99

As shown in Table 2, BRAF V600E mutation was commonly found in 3 of 5 patients with ganglion cell adenocarcinoma, and no mutation of the gene other than BRAF V600E mutation was observed. The BRAF V600E mutation confirmed in the ganglioneoclastoma is a mutation in which the 1799 thymine of the human gene BRAF gene of SEQ ID NO: 2 is substituted with adenine, and the human BRAF protein valine 600 of SEQ ID NO: 1 is substituted with glutamic acid (E) Lt; / RTI >

2-2. In enlarged patients BRAF V600E  Mutation confirmation

To confirm whether BRAF V600E, which was confirmed to exist in 5 ganglionic cell tumors in Example 2-1, was also present in other patients, the patient group was expanded to include 12 patients (5 patients in Example 2-1 GG29, GG30, GG221, GG231, and GG249) (Table 3).

Patient code
Age at surgery (years)
Address Duration of epilepsy (years)
gender Pathological finding
Radiological findings How to Sequence
BRAF V600E mutant allele ratio (%) GG29 14 Relapse
3 male
White glandular ganglion cell On the preoperative image, a 1 cm round lesion with edema was located above the right edge all - GG30 15 Intermittent spastic seizures
4 male
Ganglion glioma with multiple calcifications near the ganglioneoclastoma On preoperative MRI, a cystic lesion with increased size in the thalamus Full exome sequencing
- GG54 16 Relapse
10 female
Ganglia glioma close to ganglioneoclastoma A low-grade stage cortical tumor close to the ganglion cell tumor in the left hippocampal margin, Object panel sequencing - GG57 14 Long-term intractable epilepsy
One male
Ganglia glioma close to ganglioneoclastoma 1.4 cm lump in the mesial temporal lobe Object panel sequencing
18.50 GG163 6 Long-term intractability 0.5 male
Ganglia glioma close to ganglioneoclastoma Augmented lesion in the posterior medial temporal lobe Object panel sequencing
28.87 GG221 6 Long-term intractable epilepsy
0.5 female
Ganglia glioma close to ganglioneoclastoma 1 cm cystic lesion in left temporal lobe Full exome sequencing
15.44 GG231 7 Long-term intractable epilepsy
2 male
Ganglia glioma close to ganglioneoclastoma 1.5 cm T2-enhanced lesion in the right hook Full exome sequencing
17.86 GG249 5 Long-term intractable epilepsy
One female
Ganglia glioma close to ganglioneoclastoma T2-weighted local lesion in the right posterior temporal lobe Full exome sequencing
19.05 GG263 2 Relapse One male
Ganglia glioma close to ganglioneoclastoma T2 enhancement signal in left frontal lobe Object panel sequencing
- GG351 5 seizure One male
Tumor-related intractable epilepsy close to ganglioneoclastoma
- Object panel sequencing
- GG356 9 seizure 2 male
Tumor-related intractable epilepsy close to ganglioneoclastoma
- Object panel sequencing
- GG381 7 Long-term intractable epilepsy One male
Tumor-related intractable epilepsy close to ganglioneoclastoma
A cystic lesion of 2.8 cm in area including the hook, lateral hippocampus and hypothalamus Object panel sequencing
7.41

The BRAF V600E mutation was not found in the patient's blood and was found specifically in brain tissue samples.

As a result, as shown in Table 3, it was confirmed that BRAF V600E mutation was present in 6 out of 12 patients with gliomas (rate of genetic mutation was 50%), The ratio of BRAF V600E mutant allele to BRAF normal allele was about 7 to 30%.

2-3. Patient sampling and genomic DNA extraction

Twelve patients with intractable epilepsy due to ganglioglioma (GG) used in Example 2-2 were treated with brain tissue (1-2 g), saliva (1-2 mL) Blood (approximately 5 mL), frozen tissue and formalin fixed paraffin embedded brain tissue were obtained (Department of Pediatric Neurosurgery and Pediatric Neurology, Severance Hospital). The following kit was used to isolate the patient's brain tissue, blood, saliva, freezing and genomic DNA of formalin-fixed paraffin embedded brain tissue using the manufacturer's protocol:

(Qiagen, USA), blood: Flexigene DNA kit (Qiagen, USA), saliva: prepIT2P purification kit (DNAgenotek, USA), frozen tissue and formalin fixed paraffin embedded brain tissue: Qiamp mini FFPE DNA kit (Qiagen, USA).

2-4. ganglion Sclerotoma  Specific gene mutation sequencing

To further confirm whether a BRAF V600E mutation was present in a ganglioneoclastoma patient, out of the 12 patients identified in Example 2-2, the remaining seven patients except the five patients identified in Example 2-1 had a lead depth of Hybrid capture sequencing was performed in the following manner so as to be 100-17,700.

Genomic DNA was extracted from the above-mentioned 7 ganglionic carcinoma patients in the same manner as in Example 2-3, and hybrid capture base sequence analysis was performed thereon. For the hybrid capture sequence analysis, BRAF gene-specific probes were prepared using SureDesign online tools (Agilent Technologies). Sequencing libraries were constructed using the Agilent library preparation protocols provided by the manufacturer. Sequencing was performed on the prepared sequencing library using Hiseq2500 (illumina) so that the central lead depth was 500x. The post-sequencing data was made into a bam file that can be analyzed using the Broad Institute best practice pipeline (https://www.broadinstitute.org/gatk/).

To identify brain - specific de novo somatic mutations, we selected somatic mutations found simultaneously in two algorithms, Strelka and Mutect, among the gene sequencing results of blood and brain tissue. In addition, among the genetic mutations found in the hybrid capture sequencing results, only genetic mutations satisfying the screening criterion of three or more mutated calls with a depth of 100 or more (mapping quality of 30 or more) were selected as candidate genes for the disease.

In order to reliably eliminate the errors occurring in the sequencing process, only the case where the genetic mutation rate was 1% or more was regarded as positive, and when the hybrid capture exon sequence analysis was performed on the genome DNA of blood and brain tissue, This mutation was selected as a genetic mutation based on the positive result.

As a result, BRAF V600E mutation was not observed in saliva and blood (control) of genetic mutation-positive patients (genetic mutation rate of 1% or more) as shown in Table 4 (negative). However, BRAF V600E mutations were repeatedly detected in three patients (GG221, GG231, and GG249), and the ratio of BRAF V600E mutant allele to BRAF normal allele was 7% to 30%.

patient Saliva (presence of BRAF V600E mutation) Blood (presence of BRAF V600E mutation) BRAF V600E mutant allele ratio in brain tissue (%) GG221 - -
15.44 GG231 - -
17.86 GG249 - -
19.05

Example  3. Laser capture Cell exfoliation method  Used BRAF V600E  Identification of cell-specific presence of mutations

In order to use it in patient treatment, it is necessary to produce an appropriate prototype of the model living organism. Before making the prototype of the model living organism, it is confirmed whether the BRAF V600E mutation exists precisely in the neuron or in the cell line of the glia lineage And the following experiment was carried out.

To confirm whether the BRAF V600E mutation is present in neurons or in cell lines of the glioma lineage, the BRAF V600E mutation-confirmed GG221 and GG231 patient tissues were incubated with neuron-specific markers and glial-specific markers After immunohistochemical staining, only the cells of a specific tissue type were separated using a laser capture microscope, and the presence or absence of the BRAF V600E mutation was confirmed.

A surgical tissue block (GG221, GG231 patient tissue) identified with a BRAF V600E mutation in the ganglioneoclonus patient tissue used in Example 2 was dissolved in 4% (w / v) of freshly prepared phosphate-buffered (PB) The gelatin-embedded tissue masses were cultured overnight in 7.5% (w / v) gelatin under paraformaldehyde (fixed), 20% (w / v) buffered sucrose (cryoprotect) and 10% (w / v) sucrose / And stored at -80 ° C.

The gelatin-embedded tissue mass prepared above was rapidly immersed in 2-methylbutanes at -50 ° C, and the gelatin-embedded tissue mass was rapidly frozen. Using a frozen tissue slicer (Leica) at -20 ° C, (0.2% (w / v) gelatin and 0.2% (v / v) Triton X (w / v) gelatin solution for one hour at room temperature and incubating the frozen sections on glass slides -100 in PBS) and stained with the following antibodies: mouse monoclonal anti-NeuN (1: 200, # MAB377, Millipore) and rabbit polyclonal anti-oligodendrocyte Lineage Transcription Factor 2 (Olig2) (1: 500, AB 9610, Millipore). After three PBS washes, tissue slides were stained with secondary antibodies as follows: Alexa Fluor 555-conjugated goat antibody to mouse (1: 200 dilution; A21422, Invitrogen) or rabbit (Alexa Fluor 488-conjugated goat antibody to rabbit) (1: 200 dilution; A11008, Invitrogen). The DAPI contained in the mounting solution (P36931, Life technology) was used for nuclear staining. Fluorescence images were obtained using a Leica DMI 3000 B inverted microscope. Using a PALM MicroBeam (Carl Zeiss) microscope, only about 50 cells of NeuN-positive, Olig2-negative, NeuN-negative, and Olig2-positive cells were extracted from the stained tissues and extracted with genomic DNA using QIAamp DNA Micro Kit. The amplified PCR product was purified using a MEGAquick spin total fragment purification kit (Intron, Korea), followed by Sanger sequencing using a BioDye terminator and an automatic sequencer system (Applied Biosystems).

name primer SEQ ID NO: BRAF_LCM_F 5'-TGCTTGCTCTGATAGGAAAATG -3 ' 5 BRAF_LCM_R 5'-AGCCTCAATTCTTACCA TCCAC-3 ' 6

As a result, the BRAF V600E mutation (BRAF Chr7: 140453136 for c.1799T> A mutation) was observed in two different cells of neurons (left of FIG. 2b) Was found to be present.

As shown in FIG. 2B, the presence of the same mutation in two different cells, neurons and glia, suggests that the mutation occurred in the common ancestral cells of both neurons and glia.

Example  4. BRAF V600E  In mice expressing mutations Epilepsy  Identify the patient's phenotype

4-1. Identification of spontaneous seizures in animal model mice

Conditional floxed BRAF V600E mice capable of expressing the BRAF V600E mutation in the brain tumors confirmed in Examples 1 to 3 were prepared and the mice were mated with conditional floxed tdTomato mice to produce a timed pregnant for 14 days in the embryo Later, the plasmid gene with Cre recombinase was introduced into the uterine electroporation method to produce an animal model similar to that of the patient with ganglionic cell tumor.

Specifically, in one embodiment of the present invention, a product in which the 1910th thymine is adenine-phosphorylated in the nucleotide sequence shown in SEQ ID NO: 4 is amplified using a known site-directed mutagenesis method and inserted into the Cre-dependent LoxP sequence Transgenic mice transfected with Cre-dependent condition mutants were prepared by injection into mouse embryos. When the mouse obtained by crossing the prepared mouse and the tdTomato mouse was exposed to the 14th day of pregnancy (E14), the uterine horn was exposed 2 ug / ml of Fast Green (F7252, Sigma, USA) combined with 2-3 μg of plasmid with Cre recombinase was injected into the lateral ventricle of each embryo using a pulled glass capillary . A plasmid with Cre recombinase (pCAG-Cre-IRES2-GFP, addgene, # 26646) was used to discharge 50 V to the head of the embryo with an ECM830 eletroporator (BTX-harvard apparatus) Followed by electroporation.

The brain tissue of the plasmid electroporated mouse fetus with the Cre recombinase was removed and genotyping PCR was performed using the primers and the i-Taq ™ DNA Polymerase kit (Intron, # 25021) shown in Table 6.

name primer SEQ ID NO: LSL-Braf ^V600E Fwd 5'-CCCAGGCTCTTTATGAGAA-3 ' 7 LSL-Braf ^WT Rev 5'-AGTCAATCATCCACAGAGACCT-3 ' 8 LSL-Braf ^V600E Rev 5'-GCTTGGCTGGACGTAAACTC-3 ' 9

As a result, as shown in Fig. 3A, it was confirmed that the produced mouse expresses the BRAF V637E mutation gene (although it is a mutation of mouse BRAF V637E, but it is traditionally referred to as BRAF V600E in the same manner as human BRAF V600E, hereinafter referred to as BRAF V600E ).

In order to confirm whether electrophysiologic epilepsy appears in the brain of the model mouse, a mouse embryo obtained by electroporation of the plasmid with the Cre recombinase was born, and after 3 weeks of life, video-electroencephalography video-EEG) test. In order to analyze seizure waves by electrical signals, video EEG monitoring was carried out 3 weeks after birth. After the fetus was separated from the mother, video surveillance was performed 12 hours a day to check whether tension - epileptic seizures started. Subsequently, rats with seizures were examined for spontaneous seizures with epileptic seizures by video-electrogram monitoring over 6 hours / day for 2 days or more.

To measure the frequency of epileptic seizures and non - seizure EEG seizures, video EEG data taken from 10 to 12 hours were used. One minute of data was extracted and analyzed from this data every hour.

The frequency of seizure gait and nonspecific EEG seizures was measured by an observer who did not know the genotype of the rat. The seizure gap wave is defined as the epileptic waves of 200 ms or less at regular intervals and 2 times more amplitude than the background EEG. At least two consecutive episodes (1 to 4 Hz) And it was defined as the case where all four electrodes were observed.

1.5 mm), 측두엽 부위에 2개 (AP-2.4mm, ML

2.4 mm) 소뇌부위에 1개를 식립하여 총 5개의 전극을 식립하였다. 4일간의 회복기간을 가진 후 저녁 6시부터 새벽 2시의 시간에 마우스당 2~5일간 (하루 6시간) 뇌전도 측정을 수행하였다. 뇌전도 신호는 amplifier (GRASS model 9 EEG/Polysomnograph, GRASS technologies, USA)에 의해 증폭되었으며 pCLAMP program (Molecular Devices, USA) 또는 RHD2000 amplifier, board (Intan technoloties, USA) 및 MATLAB EEGLAB(http://sccn.ucsd.edu/eeglab)을 이용하여 상기 신호를 분석하였다.Specifically, when the mouse was wet, it was confirmed whether or not a seizure occurred through only video monitoring (> 3weeks), and then an electrode was placed for measurement of the electroencephalogram. Electrodes were located in the epidural layer. Two electrodes (AP + 2.8 mm, ML

1.5 mm), two at the temporal region (AP-2.4 mm, ML

2.4 mm) were placed in the cerebellum and five electrodes were placed. After 4 days recovery period, electroencephalogram measurement was performed for 2 to 5 days (6 hours per day) per mouse at 6:00 am to 2:00 am. The electroencephalogram signals were amplified by an amplifier (GRASS model 9 EEG / Polysomnograph, GRASS technologies, USA) and analyzed using a pCLAMP program (Molecular Devices, USA) or RHD2000 amplifier, board (Intan technoloties, USA) and MATLAB EEGLAB (http: // sccn. gt; ucsd.edu/eeglab). < / RTI >

As a result, as shown in FIG. 3B, more than 90% of the mice expressing the BRAF V600E mutant gene showed spontaneous seizures accompanied by the epileptic seizure, and the epileptic seizure exhibited a high frequency of high frequency, a high amplitude of extreme frequency, Respectively. In the mice expressing the BRAF V600E mutant gene, seizure gap waves were also observed. The mice exhibiting such spontaneous seizures exhibit a systemic tension-to-seizure seizure consisting of a tense, hepatic ataxia, similar to those occurring in patients with ganglioneocellularis. In addition, it was confirmed that the electroencephalogram of the mouse exhibited a low frequency and high frequency synchronized multifrequency, that the brain airwaves of the liver had a constant high voltage, and that the late phase had a synchronized attenuation amplitude. In contrast, mice expressing the wild-type BRAF protein did not exhibit spontaneous seizures or epilepsy.

Based on the above results, it was found that spontaneous seizures accompanied by epileptic seizures occurred in mice injected with a plasmid in which a BRAF V600E mutation gene was inserted.

4-2. In animal models, And active  Confirm

In order to electrophysiologically analyze the epileptic wave appearing in the model mouse prepared in Example 4-1, the following experiment was conducted.

Specifically, the model mouse prepared in Example 4-1 was used as a vibratome to obtain a cerebral cortical slice, and spontaneous action potentials were measured in an artificial cerebrospinal fluid solution. The composition of artificial CSF was as follows: in mM, 124 NaCl, 26 NaHCO3, 3 KCl, 1.25 KH2PO4, 2 CaCl2, 1 MgSO4, and 10 D-glucose. The mouse brain cortical slice was supported on a brain multi-channel electrode recording device (MED64 probe, # P515A, Panasonic Alpha-Med Sciences) using a slide anchor kit (SHD-22CKIT, Warner Instruments) The epileptogenesis was measured at 37 ° C / 5% CO 2 while flowing artificial cerebrospinal fluid at a rate of 2 mL / min. Spikes were detected using Mobius software (Alpha Med Scientific).

 The results are shown in FIG. 3C and FIG.

On the left side of FIG. 3c, 90% of the mice had epileptic seizures when BRAF V600E mutation was induced, and in the middle of FIG. 3c, the seizure- .

On the right side of FIG. 3c, we can see when the individual seizures of BRAF V600E mutant mice causing seizures start in FIG. 3c.

FIG. 4 shows that, unlike the control group, a synchronized burst firing occurs in brain tissue with a BRAF V600E mutation, which is characterized by spontaneous activity waves and temporally short periods of high amplitude energy simultaneously in several channels.

Therefore, unlike brain tissues obtained from mice expressing the wild type BRAF protein as a control group, the brain tissue obtained from the mice expressing the BRAF V600E mutant protein exhibited spontaneous activity waves characteristic of brain metastasis, The synchronized burst firing in which the high amplitude energy of the cycle is emitted appears. As a result, it was confirmed that the neuron was overactivated in the model mouse prepared in Example 4-1.

Example  5: BRAF V600E  Of mutant mice Epilepsy  Identification of association tumor specificity

5-1. BRAF V600E  Mutant animal model mouse Immunofluorescent staining

The brain tissue of the animal model mouse prepared in Example 4-1 was tested in the same manner as in Example 3 to prepare a gelatin-embedded tissue mass, which was stored at -80 ° C.

The gelatin-embedded tissue mass was frozen sectioned on the glass slide and stained with the following antibodies: mouse monoclonal anti-NeuN (1: 200, # MAB377, Millipore), rabbit polyclonal anti- glial fibrillary acidic protein (GFAP) (1: 500, # z0334, DAKO), rabbit polyclonal anti-oligodendrocyte Lineage Transcription Factor 2 (Olig2) (1: 500, AB9610, Millipore), rabbit monoclonal anti- , monoclonal anti-parvalbumin (PV) monoclonal anti-CD34 (1: 500, ab81289, Abcam), mouse monoclonal anti-glutamate decarboxylase 67 (GAD67) (1: 500, # MAB5406, Millipore) (1: 500, # 135 303, Synaptic Systems), rabbit polyclonal anti-vesicular GABA transporter (VGAT) (1: 500, # MAB1572, Millipore), rabbit polyclonal anti-vesicular glutamate transporter 1 # 135 002, Synaptic Systems), mouse monoclonal anti-GFP (1: 500, # Ab1218, Abcam), rabbit polyclonal anti-GFP (1: 500, # Ab290, Abcam) polyclonal anti-REST (1: 200, IHC-00141, Bethyl Laboratories) and rabbit polyclonal anti-CUX1 (1: 400, SC13024, Santa Cruz). After three PBS washes, tissue slides were stained with secondary antibody: Alexa 488-conjugated goat anti-rabbit IgG (1: 500, # A11008, Invitrogen) or Alexa 555-conjugated goat anti-rabbit IgG , # 21428, Invitrogen). The DAPI contained in the mounting solution (P36931, Life technology) was used for nuclear staining. Images were acquired using a Leica DMI 3000 B inverted microscope or a Zeiss LSM780 confocal microscope. NeuN-positive cell numbers were measured using a 10x objective lens; Within the neuron-rich regina, 4 to 5 fields per subject were obtained, and more than 100 cells per region were recorded. The number of DAPI-positive cells represents the total number of cells. Neuron cell size was measured in NeuN-positive cells using ImageJ software's automated counting protocol of ImageJ software (http://rsbweb.nih.gov/ij/). The circularity and aspect ratio of the neurons and the angles to the cortical surface of the dendrites were automatically calculated by ImageJ.

5-2. Immunofluorescent stained  Identification of neuronal dysplasia by quantitative analysis of tissue

The results of immunofluorescence staining in the same manner as in Example 5-1 are shown in Fig.

As a result, as shown in FIG. 5, in the case of GFP-positive cells of the cerebral region induced by electroporation of the BRAF V600E mutation in the same manner as in Example 4, neuronal dysplasia characteristic of glioblastoma is accompanied (FIG. 5A). In the case of the GFP-positive cells, the size of the cells became larger and the shape of the cells became distorted (FIG. 5B) (C in Fig. 5) that are arranged in an arbitrary direction.

5-3. Immunofluorescent stained  Through quantitative analysis of tissue Giant cell  Confirmation of increased proliferation

In order to track daughter cells differentiated in neural source cells with BRAF V600E mutations in animal model mice prepared using the same method as in Example 4-1, the same method as in Example 4-1 was used to determine the Cre recombinase-dependent A transgenic mouse substituted with a gene expressing BRAF V600E protein was crossed with a mouse capable of producing a tdTomato fluorescent protein to obtain a mouse, and the Cre recombinant enzyme was introduced into the mouse by the uterine electroporation method, Daughter cells differentiated from BRAF V600E mutant neurogenic cells were traced in tdTomato fluorescent color.

As a result, as shown in Fig. 6b, daughter cells differentiated from BRAF V600E mutant neuronal cytoskeleton were found to have BRAF normal (Fig. 6B) Was significantly increased compared to the brain tissue of the mouse expressing the gene.

Taken together, the results of Examples 5-2 and 5-3 show that the BRAF V600E mutant prepared in Example 4 is an animal model that reflects neuronal dysplasia and ganglion cell hyperplasia, Able to know.

5-4. Immunofluorescent stained  CD34 through quantitative analysis of tissue Marker  Confirm

Since ganglioneoclastoma is known to have an increased pathologically expressing amount of CD34 in tumor tissue, it has been known that an animal model mouse tissue prepared in the same manner as in Example 4-1 can be used in the same manner as in Example 5-1 Immunofluorescent staining was performed to confirm the expression level of CD34 marker.

As a result, as shown in FIG. 7, it was confirmed that the expression of CD34 was 2.2-fold increased in the animal model mouse tissue prepared in the same manner as in Example 4-1, unlike the control group.

In order to further confirm the features of the renal tuberculoma, hematoxylin-eosin and DAB immunohistochemical staining used for pathologically diagnosing the renal tuberculoma were carried out in the same manner as in Example 4-1 Animal models Mouse tissues were stained.

Specifically, formalin (Sigma, # HT501128) was injected and fixed through the heart of an animal model mouse prepared in the same manner as in Example 4-1, and paraffin was infiltrated into a paraffin foraging machine (Leica TP1020) Fixed paraffin embedded mouse brain tissue was obtained. The brain tissue of the formalin-fixed paraffin embedded mouse was cut into a 4-μm-thick microtome (Leica RM 2135), and the slide was subjected to general hematoxylin-eosin staining. For the immunohistochemical staining of CD34, the slides were firstly collected with sodium citrate, dehydrated with a gradual concentration of alcohol, and then diluted with 3% (w / w) H2O2 (Sigma aldrich, H6520) The intrinsic peroxidase activity was removed. Tissues were blocked with blocking solution of PBS-GT (0.2% (w / v) gelatin and 0.2% (v / v) Triton X-100 under PBS) and then incubated with anti-CD34 primary antibody (1: 500, ab81289, Abcam) at room temperature. After washing three times with PBS, the cells were stained with a secondary antibody. The antigen binding sites in the tissues were visualized using 3,3'-Diaminobenzidine substrate solution (DAB, Vector Laboratories). To rule out bias due to image intensity, both normal and BRAF V600E mutant mouse tissues were reacted for the same time and background staining was performed with hematoxylin.

As a result, as shown in the right picture of FIG. 8, it was confirmed that CD34 expression, which was not stained in normal tissues, was remarkably expressed in BRAF V600E mutant mouse brain tissue in the area where mutation occurred.

7 to 8, the expression of CD34 was remarkably increased in mouse brain tissues with a BRAF V600E somatic mutation as compared with that of normal brain mouse tissues, And the nerve cells and tissues induced by the mutation.

5-5. Immunofluorescent stained  Through quantitative analysis of tissue Lamellar  Confirm

It is known that ganglioneoclastomas are often accompanied by cortical dysplasia in the tumor tissue or by cortical dyslamination as a part of developmental anomaly of the cortex. Therefore, the animal model prepared in the same manner as in Example 4-1 In order to confirm whether or not a similar pattern appears in the mouse tissues, the animal model mouse tissues prepared in the same manner as in Example 4-1 were fixed and prepared in the same manner as in Example 5-4, Dyeing was carried out through the Cux1 marker which can be identified by dividing the layers.

As a result, as shown in FIG. 9, it was confirmed that Cux1 marker was strongly observed not only in the upper part of the cortex but also in the lower part in mouse brain tissue having a BRAF V600E somatic mutation, unlike a mouse brain tissue having a normal gene. In particular, the distribution of Cux1-positive neurons was significantly negative in BRAF V600E mutant mice, indicating that abnormal lamination of mouse cerebral cortex with BRAF V600E somatic mutation was evident .

In addition, the animal model mouse tissue prepared in the same manner as in Example 4-1 was subjected to immunohistochemical fluorescence analysis in the same manner as in Example 5-1, and the distribution of cells simultaneously positive to NeuN and tdTomato was confirmed. As can be seen, NeuN-positive and tdTomato-positive neurons in the lower cortical layer, where cells derived from mouse brain tissue with the BRAF V600E mutation were not normally distributed, contained a significant amount of the protein (r = -0.1122, p <0.0001 )).

However, it can be seen from the bottom of FIG. 10 that the hyperplasia observed in the glioblastoma shown in Example 5-3 is not simultaneously transmitted to the neuron. In other words, in neurons, there was no cell proliferation and only the location in the cortex was present.

5-6. Summary of results "G Summary

The results of Examples 5-2, 5-3, 5-4, and 5-5 show that mouse model tissues produced in the same manner as in Example 4-1 were classified into brain metastases And the findings of the tumors with high incidence of symp- toms were similar. These findings include neuronal dysplasia due to BRAF V600E mutation, hypercellular hyperplasia, abnormal arrangement of neurite dendrites, CD34 positive findings, and cortical abnormalities.

Example  6. BRAF V600E  Phenotype by spatial acquisition of mutation

As described in Examples 3 and 5-2, the animal model mice presented in the present invention have a BRAF V600E mutation in their glioblastoma cells, It is necessary to confirm whether any mutation in the derived mutation plays an important role in inducing brain metastasis.

For this purpose, a mouse capable of expressing Cre-dependent expression of the BRAF V600E mutation found in a ganglionic cell tumor patient was prepared in a manner similar to Example 4-1, and a plasmid having a Cre recombinase gene was expressed on the first day after the mouse birth After the puncture, the behavior was observed for about 90 days.

Specifically, the differentiation of the cortical nervous system involves the development of neurons at E14, and the differentiation of cells at birth. Therefore, the Cre plasmid is injected into the embryo capable of expressing Cre- Mutation occurs in neurons and glial cells, and a Cre plasmid is injected at the time of the first day after birth of the mouse. Therefore, in order to generate the BRAF V600E mutation only in the diplopia using this fact, 4-1, mice were injected with Cre plasmid at the first day after birth, in which Cre-dependent gene expression was possible.

As a result, as shown in Fig. 11B, it was confirmed that no seizure occurred in mice (GFAP and OLIG2 glioma confirmation markers) which were produced so as to generate a BRAF V600E mutation only in the glia.

In addition, in order to confirm whether or not the glial hyperplasia also occurs in the mouse produced to produce the BRAF V600E mutation only in the glial cell, the experiment as in Example 5-3 showed that the glial hyperplasia remained significantly as shown in Fig. 11C .

In conclusion, we concluded that BRAF V600E mutation in BRAF V600E mutation plays an important role in the development of epilepsy. BRAF V600E mutation plays an important role in the pathogenesis of epilepsy. In addition, it was found that the proliferation of benign tumors in which BRFA V600E mutation occurs only in the diplopia is increased.

Example  7: BRAF V600E  Phenotype by temporal acquisition of mutation

As the epidemiologic information of the patient is found at a high rate in pediatric patients, the present inventive mouse model also shows whether there is a phenotypic difference in acquisition of BRAF V600E mutation in childhood and mutation acquisition in adult mouse And confirmed by the following method.

7-1. Using Tamoxifen in adult mice Inducible BRAF V600E  Mutation

In order to change the timing of BRAF V600E mutation on an animal model mouse, the following method was used.

Timed pregnant mice of conditional floxed BRAF V600E mice were prepared in the same manner as in Example 4-1, and a plasmid gene with tamoxifen inducible Cre recombinase was intrauterine transfused. After 30 days from birth, tamoxifen was intraperitoneally injected BRAF V600E somatic mutation was appropriately induced in the model mouse brain. Tamoxifen began to be treated 30 days after birth and the behavior of the mice was monitored for about 30 days.

Specifically, intrauterine inheritance was induced by exposing the uterine horn of Timed pregnant 14 days (E14) and binding to 2 to 3 ug of plasmid with Cre recombinase in the lateral ventricle of each embryo 2 ug / ml of Fast Green (F7252, Sigma, USA) was injected using pulled glass capillary. The plasmid was electroporated by discharging 50 V with an ECM 830 eletroporator (BTX-harvard apparatus), which was an electric pulse five times 100 ms at intervals of 900 ms at the head of the embryo. In addition, tamoxifen (Sigma, T5648) was kept in a dark condition at a concentration of 10 mg / ml in corn oil (Sigma, C8267) at 37 ° C for one day and 100 ug / g of tamoxifen was intraperitoneally injected into both normal and mutant mice Five days were consecutively administered once a day, followed by one week immediately after the absence period, and then five consecutive days. After treatment with tamoxifen, the mice began to observe behavior, and mice treated with either tamoxifen-free corn oil or tamoxifen-treated mice were used as controls and their behavior was compared. A schematic flow of the above experimental procedure is shown in FIG.

As a result, as shown in the graph on the right of FIG. 12, when the BRAF V600E mutation was induced in the neurons of adult mice after 30 days of age, the mouse did not exhibit seizure activity.

7-2. Virus-induced BRAF V600E  Mutant mouse

In order to change the timing of BRAF V600E mutation on an animal model mouse, BRAF V600E mutant mice were prepared by the following method using virus.

Specifically, mice 30 days after birth were anesthetized, the head fixed in a stereotaxic surgical apparatus (Stoelting Co., Wood Dale, Ill.), And a hole having a diameter of about 1 mm was made into a dental drill in a brain bone. 0.5 μL of AAV9.CamKII.HI.eGFP-Cre.WPRE.SV40 (Penn Vector Core Philadelphia, PA; titer 6.54 x 1013 GCml-1) was diluted with a somatosensory cortex (AP: -0.5; ~ -2.5; DV: -0.5) at a slow rate (1 nL per sec). Two weeks later, the mice were observed for the virus-injected mice and the brain waves were measured. After observation, the brain was prepared in the same manner as in Example 5-1, and the frozen tissue slides were prepared with a 20-μm-thick paraformaldehyde and observed under a microscope to confirm whether the fluorescent reporter protein injected through the virus was well expressed Respectively.

As a result, as shown in Fig. 13, as in the case of Example 7-1, a BRAF V600E mutation was induced in adult mice using tamoxifen, and no mutation was induced in the mice.

7-3. From an adult mouse BRAF V600E  Cytological abnormalities induced by mutation induction

After the BRAF V600E somatic mutation was induced in adult mice as in the methods of Examples 7-2 and 7-3, neuronal dysfunction was confirmed by the same method as in Example 5-2.

As a result, as shown in Fig. 14, when BRAF V600E mutation was induced in adult mice (BRAFWT / LSL-V600E), as in BRAF V600E mutant mice prepared in the same manner as in Example 4-1, normal BRAF gene (Fig. 14A), cell circularity, aspect ratio (Fig. 14B) and angles of the dendrites with respect to the cortical surface (Fig. 14C) compared with the mouse BRAFWT / There was no significant difference between the mice bearing the normal BRAF gene (no neuronal dysplasia).

In addition, when BRAF V600E mutation was induced in adult mice using tamoxifen, the size of the cell, circularity, aspect ratio, and angle of the dendrite to the cortical surface were compared with those of the normal BRAF gene, (Fig. 14D) (absence of neuronal dysplasia).

These results indicate that the BRAF V600E mutation induces neuronal dysplasia and induces brain metastasis when it occurs only in neurons in embryonic developmental stage.

Example  8. BRAF V600E  Identification of seizure reduction through injection of specific inhibitor

The BRAF V600E-specific inhibitor was treated and its effect was confirmed in order to reduce the incidence of seizures in the BRAF V600E mutant mouse produced in the same manner as in Example 4-1.

Specifically, a long-term BRAF V600E mutant protein-specific activity inhibitor (Vemurafenib) was injected into brain tissue of a BRAF V600E mutant mouse produced in the same manner as in Example 4-1. A total of 30 patients were treated with an intramuscular injection of zoletil (0.01 mg / kg) and rompun (0.2 mg / kg) and an osmotic pump (ALZET pumps 2004 or 2006, ALZET Osmotic Vehicle (50% (v / v) DMSO on DW) or 500 uM PLX4032 (V-2800, LC laboratories) were placed in the right ventricular space (AP: -0.6; ML: -1.0; DV: -2.0) and chronic intracranioventricular injection (cICV). Four EEG probes (left / right frontal lobes and left temporal lobes, cerebellum as a reference) were inserted into the head of an animal model mouse in the same manner as in Example 4-1, After ~ 6 weeks, the mice were sacrificed and the brain was taken out for further study. At this time, we confirmed whether the semipermeable membrane in the osmotic pump was reduced. Drug infusion was performed from 4 weeks to 6 weeks, at which point the mouse brain waves were tracked.

The results are shown in Fig. 15 to Fig.

15, it was confirmed that mice injected with a BRAF V600E-specific inhibitor decreased the icta seizure by about 4 times (Fig. 15B), and even when treated with the BRAF V600E-specific inhibitor for 6 weeks, (Fig. 15C).

16, it was confirmed that the electrophysiological seizure was reduced by 2 to 3 times (in the middle of FIG. 16), and the interictal spike was reduced by 1.3 to 1.5 times.

17, a BRAF V600E specific inhibitor is temporarily injected into the ventricle (3 times / day, 5 uL for 8 minutes) (Fig. 17, left) or oral administration (PO, per oral) (Fig. 17, right), it was confirmed that the seizure frequency did not decrease.

Example  9. BRAF V600E  Mechanisms associated with seizures by mutation

The abnormal activity of the mTOR signaling pathway is known to induce epileptic seizures, and the BRAF V600E mutation is known to activate the MAPK signaling pathway of Ras-Raf-MEK-ERK. On the other hand, the MAPK signaling pathway of Ras-Raf-MEK-ERK, which is known to activate the BRAF V600E mutation, is known to activate the mTOR signaling pathway by crosstalking with the mTOR signaling pathway, Have been reported to increase the expression of proteins associated with the mTOR signaling pathway. Therefore, the following experiment was conducted to confirm whether the mTOR signaling pathway was activated by the BRAF V600E mutation to cause epileptic seizure.

Specifically, rapamycin, an mTOR-specific inhibitor, was injected into an animal model mouse prepared in the same manner as in Example 4-1 by intraperitoneal injection, and the change was confirmed. Rapamycin (LC Labs, USA) was diluted to 20 mg / ml in 100% ethanol and the stock solution was stored at -20 ° C. Rapamycin was diluted in 5% (w / v) polyethleneglycol 400 and 5% (v / v) Tween 80 before being injected into animal model mice. Solution. The prepared solution was administered by intraperitoneal injection at a concentration of 1 to 10 mg / kg for 2 weeks (10 mg / kg / d ip, for 2 weeks).

As a result, as shown in Fig. 18, the frequency of epileptic seizures in BRAF V600E mutant animal model mice was not reduced despite the administration of rapamycin, and the phosphorylation of S6 protein in the mTOR subpathway was significantly lower than that of BRAF V600E mutant animal There was no significant difference in model mouse tissue.

<110> KOREA ADVANCED INSTITUTE OF SCIENCE AND TECHNOLOGY          YONSEI UNIVERSITY, UNIVERSITY? INDUSTRY FOUNDATION (UIF) <120> Epilepsy animal modeling using somatic BRAF mutation and its          application for drug screening <130> DPP-2017-2502-KR <160> 9 <170> KoPatentin 3.0 <210> 1 <211> 766 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ala Leu Ser Gly Gly Gly Gly Gly Gly Gly Ala Glu Pro Gly Gln   1 5 10 15 Ala Leu Phe Asn Gly Asp Met Glu Pro Glu Ala Gly Ala Gly Ala Gly              20 25 30 Ala Ala Ala Ser Ser Ala Ala Asp Pro Ala Ile Pro Glu Glu Val Trp          35 40 45 Asn Ile Lys Gln Met Ile Lys Leu Thr Gln Glu His Ile Glu Ala Leu      50 55 60 Leu Asp Lys Phe Gly Gly Glu His Asn Pro Pro Ser Ile Tyr Leu Glu  65 70 75 80 Ala Tyr Glu Glu Tyr Thr Ser Lys Leu Asp Ala Leu Gln Gln Arg Glu                  85 90 95 Gln Gln Leu Leu Glu Ser Leu Gly Asn Gly Thr Asp Phe Ser Val Ser             100 105 110 Ser Ser Ala Ser Met Ser Ser Ser Ser         115 120 125 Ser Val Leu Pro Ser Ser Leu Ser Val Phe Gln Asn Pro Thr Asp Val     130 135 140 Ala Arg Ser Asn Pro Lys Ser Pro Gln Lys Pro Ile Val Arg Val Phe 145 150 155 160 Leu Pro Asn Lys Gln Arg Thr Val Val Pro Ala Arg Cys Gly Val Thr                 165 170 175 Val Arg Asp Ser Leu Lys Lys Ala Leu Met Met Arg Gly Leu Ile Pro             180 185 190 Glu Cys Cys Ala Val Tyr Arg Ile Gln Asp Gly Glu Lys Lys Pro Ile         195 200 205 Gly Trp Asp Thr Asp Ile Ser Trp Leu Thr Gly Glu Glu Leu His Val     210 215 220 Glu Val Leu Glu Asn Val Pro Leu Thr Thr His Asn Phe Val Arg Lys 225 230 235 240 Thr Phe Phe Thr Leu Ala Phe Cys Asp Phe Cys Arg Lys Leu Leu Phe                 245 250 255 Gln Gly Phe Arg Cys Gln Thr Cys Gly Tyr Lys Phe His Gln Arg Cys             260 265 270 Ser Thr Glu Val Pro Leu Met Cys Val Asn Tyr Asp Gln Leu Asp Leu         275 280 285 Leu Phe Val Ser Lys Phe Phe Glu His His Pro Ile Pro Gln Glu Glu     290 295 300 Ala Ser Leu Ala Glu Thr Ala Leu Thr Ser Gly Ser Ser Ser Ala 305 310 315 320 Pro Ala Ser Asp Ser Ile Gly Pro Gln Ile Leu Thr Ser Pro Ser Pro                 325 330 335 Ser Lys Ser Ile Pro Ile Pro Gln Pro Phe Arg Pro Ala Asp Glu Asp             340 345 350 His Arg Asn Gln Phe Gly Gln Arg Asp Arg Ser Ser Ser Ala Pro Asn         355 360 365 Val His Ile Asn Thr Ile Glu Pro Val Asn Ile Asp Asp Leu Ile Arg     370 375 380 Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr Thr Gly Leu Ser Ala 385 390 395 400 Thr Pro Pro Ala Ser Leu Pro Gly Ser Leu Thr Asn Val Lys Ala Leu                 405 410 415 Gln Lys Ser Pro Gly Pro Gln Arg Glu Arg Lys Ser Ser Ser Ser             420 425 430 Glu Asp Arg Asn Arg Met Lys Thr Leu Gly Arg Arg Asp Ser Ser Asp         435 440 445 Asp Trp Glu Ile Pro Asp Gly Gln Ile Thr Val Gly Gln Arg Ile Gly     450 455 460 Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val 465 470 475 480 Ala Val Lys Met Leu Asn Val Thr Ala Pro Thr Pro Gln Gln Leu Gln                 485 490 495 Ala Phe Lys Asn Glu Val Gly Val Leu Arg Lys Thr Arg His Val Asn             500 505 510 Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys Pro Gln Leu Ala Ile Val         515 520 525 Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr His His Leu His Ile Ile     530 535 540 Glu Thr Lys Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Arg Gln Thr 545 550 555 560 Ala Gln Gly Met Asp Tyr Leu His Ala Lys Ser Ile Ile His Arg Asp                 565 570 575 Leu Lys Ser Asn Asn Ile Phe Leu His Glu Asp Leu Thr Val Lys Ile             580 585 590 Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His         595 600 605 Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pro Glu Val     610 615 620 Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp Val Tyr 625 630 635 640 Ala Phe Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Gln Leu Pro Tyr                 645 650 655 Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly             660 665 670 Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pro Lys Ala         675 680 685 Met Lys Arg Leu Met Ala Glu Cys Leu Lys Lys Lys Arg Asp Glu Arg     690 695 700 Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile Glu Leu Leu Ala Arg Ser 705 710 715 720 Leu Pro Lys Ile His Arg Ser Ala Ser Glu Pro Ser Leu Asn Arg Ala                 725 730 735 Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser Pro Lys             740 745 750 Thr Pro Ile Gln Ala Gly Gly Tyr Gly Ala Phe Pro Val His         755 760 765 <210> 2 <211> 2949 <212> DNA <213> Homo sapiens <400> 2 cgcctccctt ccccctcccc gcccgacagc ggccgctcgg gccccggctc tcggttataa 60 gatggcggcg ctgagcggtg gcggtggtgg cggcgcggag ccgggccagg ctctgttcaa 120 cggggacatg gagcccgagg ccggcgccgg cgccggcgcc gcggcctctt cggctgcgga 180 ccctgccatt ccggaggagg tgtggaatat caaacaaatg attaagttga cacaggaaca 240 tatagaggcc ctattggaca aatttggtgg ggagcataat ccaccatcaa tatatctgga 300 ggcctatgaa gaatacacca gcaagctaga tgcactccaa caaagagaac aacagttatt 360 ggaatctctg gggaacggaa ctgatttttc tgtttctagc tctgcatcaa tggataccgt 420 tacatcttct tcctcttcta gcctttcagt gctaccttca tctctttcag tttttcaaaa 480 tcccacagat gtggcacgga gcaaccccaa gtcaccacaa aaacctatcg ttagagtctt 540 cctgcccaac aaacagagga cagtggtacc tgcaaggtgt ggagttacag tccgagacag 600 tctaaagaaa gcactgatga tgagaggtct aatcccagag tgctgtgctg tttacagaat 660 tcaggatgga gagaagaaac caattggttg ggacactgat atttcctggc ttactggaga 720 agaattgcat gtggaagtgt tggagaatgt tccacttaca acacacaact ttgtacgaaa 780 aacgtttttc accttagcat tttgtgactt ttgtcgaaag ctgcttttcc agggtttccg 840 ctgtcaaaca tgtggttata aatttcacca gcgttgtagt acagaagttc cactgatgtg 900 tgttaattat gaccaacttg atttgctgtt tgtctccaag ttctttgaac accacccaat 960 accacaggaa gaggcgtcct tagcagagac tgccctaaca tctggatcat ccccttccgc 1020 acccgcctcg gactctattg ggccccaaat tctcaccagt ccgtctcctt caaaatccat 1080 tccaattcca cagcccttcc gaccagcaga tgaagatcat cgaaatcaat ttgggcaacg 1140 agaccgatcc tcatcagctc ccaatgtgca tataaacaca atagaacctg tcaatattga 1200 tgacttgatt agagaccaag gatttcgtgg tgatggagga tcaaccacag gtttgtctgc 1260 taccccccct gcctcattac ctggctcact aactaacgtg aaagccttac agaaatctcc 1320 aggacctcag cgagaaagga agtcatcttc atcctcagaa gacaggaatc gaatgaaaac 1380 acttggtaga cgggactcga gtgatgattg ggagattcct gatgggcaga ttacagtggg 1440 acaaagaatt ggatctggat catttggaac agtctacaag ggaaagtggc atggtgatgt 1500 ggcagtgaaa atgttgaatg tgacagcacc tacacctcag cagttacaag ccttcaaaaa 1560 tgaagtagga gtactcagga aaacacgaca tgtgaatatc ctactcttca tgggctattc 1620 cacaaagcca caactggcta ttgttaccca gtggtgtgag ggctccagct tgtatcacca 1680 tctccatatc attgagacca aatttgagat gatcaaactt atagatattg cacgacagac 1740 tgcacagggc atggattact tacacgccaa gtcaatcatc cacagagacc tcaagagtta 1800 taatatattt cttcatgaag acctcacagt aaaaataggt gattttggtc tagctacagt 1860 gaaatctcga tggagtgggt cccatcagtt tgaacagttg tctggatcca ttttgtggat 1920 ggcaccagaa gtcatcagaa tgcaagataa aaatccatac agctttcagt cagatgtata 1980 tgcatttgga attgttctgt atgaattgat gactggacag ttaccttatt caaacatcaa 2040 caacagggac cagataattt ttatggtggg acgaggatac ctgtctccag atctcagtaa 2100 ggtacggagt aactgtccaa aagccatgaa gagattaatg gcagagtgcc tcaaaaagaa 2160 aagagatgag agaccactct ttccccaaat tctcgcctct attgagctgc tggcccgctc 2220 attgccaaaa attcaccgca gtgcatcaga accctccttg aatcgggctg gtttccaaac 2280 agaggatttt agtctatatg cttgtgcttc tccaaaaaca cccatccagg cagggggata 2340 tggtgcgttt cctgtccact gaaacaaatg agtgagagag ttcaggagag tagcaacaaa 2400 aggaaaataa atgaacatat gtttgcttat atgttaaatt gaataaaata ctctcttttt 2460 ttttaaggtg aaccaaagaa cacttgtgtg gttaaagact agatataatt tttccccaaa 2520 ctaaaattta tacttaacat tggattttta acatccaagg gttaaaatac atagacattg 2580 ctaaaaattg gcagagcctc ttctagaggc tttactttct gttccgggtt tgtatcattc 2640 acttggttat tttaagtagt aaacttcagt ttctcatgca acttttgttg ccagctatca 2700 catgtccact agggactcca gaagaagacc ctacctatgc ctgtgtttgc aggtgagaag 2760 ttggcagtcg gttagcctgg gttagataag gcaaactgaa cagatctaat ttaggaagtc 2820 agtagaattt aataattcta ttattattct taataatttt tctataacta tttcttttta 2880 taacaatttg gaaaatgtgg atgtctttta tttccttgaa gcaataaact aagtttcttt 2940 ttataaaaa 2949 <210> 3 <211> 804 <212> PRT <213> Mus musculus <400> 3 Met Ala Ala Leu Ser Gly Gly Gly Gly Ser Ser Ser Gly Gly Gly Gly   1 5 10 15 Gly Gly Gly Gly Gly Gly Gly Gly Asp Gly Gly Gly Gly Ala Glu              20 25 30 Gln Gly Gln Ala Leu Phe Asn Gly Asp Met Glu Pro Glu Ala Gly Ala          35 40 45 Gly Ala Ala Ala Ser Ser Ala Ala Asp Pro Ala Ile Pro Glu Glu Val      50 55 60 Trp Asn Ile Lys Gln Met Ile Lys Leu Thr Gln Glu His Ile Glu Ala  65 70 75 80 Leu Leu Asp Lys Phe Gly Gly Glu His Asn Pro Pro Ser Ile Tyr Leu                  85 90 95 Glu Ala Tyr Glu Glu Tyr Thr Ser Lys Leu Asp Ala Leu Gln Gln Arg             100 105 110 Glu Gln Gln Leu Leu Glu Ser Leu Val Phe Gln Thr Pro Thr Asp Ala         115 120 125 Ser Arg Asn Asn Pro Lys Ser Pro Gln Lys Pro Ile Val Arg Val Phe     130 135 140 Leu Pro Asn Lys Gln Arg Thr Val Val Pro Ala Arg Cys Gly Val Thr 145 150 155 160 Val Arg Asp Ser Leu Lys Lys Ala Leu Met Met Arg Gly Leu Ile Pro                 165 170 175 Glu Cys Cys Ala Val Tyr Arg Ile Gln Asp Gly Glu Lys Lys Pro Ile             180 185 190 Gly Trp Asp Thr Asp Ile Ser Trp Leu Thr Gly Glu Glu Leu His Val         195 200 205 Glu Val Leu Glu Asn Val Pro Leu Thr Thr His Asn Phe Val Arg Lys     210 215 220 Thr Phe Phe Thr Leu Ala Phe Cys Asp Phe Cys Arg Lys Leu Leu Phe 225 230 235 240 Gln Gly Phe Arg Cys Gln Thr Cys Gly Tyr Lys Phe His Gln Arg Cys                 245 250 255 Ser Thr Glu Val Pro Leu Met Cys Val Asn Tyr Asp Gln Leu Asp Leu             260 265 270 Leu Phe Val Ser Lys Phe Phe Glu His His Pro Val Gln Glu Glu         275 280 285 Ala Ser Phe Pro Glu Thr Ala Leu Pro Ser Ser Ser Ser Ser Ala Pro     290 295 300 Pro Ser Asp Ser Thr Gly Pro Gln Ile Leu Thr Ser Ser Ser Ser Ser 305 310 315 320 Lys Ser Ile Pro Ile Pro Gln Pro Phe Arg Pro Ala Asp Glu Asp His                 325 330 335 Arg Asn Gln Phe Gly Gln Arg Asp Arg Ser Ser Ser Ala Pro Asn Val             340 345 350 His Ile Asn Thr Ile Glu Pro Val Asn Ile Asp Glu Lys Phe Pro Glu         355 360 365 Val Glu Leu Gln Asp Gln Arg Asp Leu Ile Arg Asp Gln Gly Phe Arg     370 375 380 Gly Asp Gly Ala Pro Leu Asn Gln Leu Met Arg Cys Leu Arg Lys Tyr 385 390 395 400 Gln Ser Arg Thr Ser Ser Pro Leu Leu His Ser Val Ser Ser Glu Ile                 405 410 415 Val Phe Asp Phe Glu Pro Gly Pro Val Phe Arg Gly Ser Thr Thr Gly             420 425 430 Leu Ser Ala Thr Pro Pro Ala Ser Leu Pro Gly Ser Leu Thr Asn Val         435 440 445 Lys Ala Leu Gln Lys Ser Pro Gly Pro Gln Arg Glu Arg Lys Ser Ser     450 455 460 Ser Ser Ser Ser Glu Asp Arg Ser Arg Met Lys Thr Leu Gly Arg 465 470 475 480 Arg Asp Ser Ser Asp Asp Trp Glu Ile Pro Asp Gly Gin Ile Thr Val                 485 490 495 Gly Gln Arg Ile Gly Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys             500 505 510 Trp His Gly Asp Val Ala Val Lys Met Leu Asn Val Thr Ala Pro Thr         515 520 525 Pro Gln Gln Leu Gln Ala Phe Lys Asn Glu Val Gly Val Leu Arg Lys     530 535 540 Thr Arg His Val Asn Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys Pro 545 550 555 560 Gln Leu Ala Ile Val Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr His                 565 570 575 His Leu His Ile Ile Glu Thr Lys Phe Glu Met Ile Lys Leu Ile Asp             580 585 590 Ile Ala Arg Gln Thr Ala Gln Gly Met Asp Tyr Leu His Ala Lys Ser         595 600 605 Ile Ile His Arg Asp Leu Lys Ser Asn Asn Ile Phe Leu His Glu Asp     610 615 620 Leu Thr Val Lys Ile Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg 625 630 635 640 Trp Ser Gly Ser His Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu Trp                 645 650 655 Met Ala Pro Glu Val Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser Phe             660 665 670 Gln Ser Asp Val Tyr Ala Phe Gly Ile Val Leu Tyr Glu Leu Met Thr         675 680 685 Gly Gln Leu Pro Tyr Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile Phe     690 695 700 Met Val Gly Arg Gly Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg Ser 705 710 715 720 Asn Cys Pro Lys Ala Met Lys Arg Leu Met Ala Glu Cys Leu Lys Lys                 725 730 735 Lys Arg Asp Glu Arg Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile Glu             740 745 750 Leu Leu Ala Arg Ser Leu Pro Lys Ile His Arg Ser Ala Ser Glu Pro         755 760 765 Ser Leu Asn Arg Ala Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr Ala     770 775 780 Cys Ala Ser Pro Lys Thr Pro Ile Gln Ala Gly Gly Tyr Gly Glu Phe 785 790 795 800 Ala Ala Phe Lys                 <210> 4 <211> 9728 <212> DNA <213> Mus musculus <400> 4 ccctcaggct cggctgcgcc ggggccgccg gcgggttcca gaggtggcct ccgccccggc 60 cgctccgccc acgccccccg cgcctccgcg cccgcctccg cccgccctgc gcctcccttc 120 cccctccccg ccccgcggcg gccgctcggc ccggctcgcg cttcgaagat ggcggcgctg 180 cggtggcggt ggcgacggcg gcggcggcgc cgagcagggc caggctctgt tcaatggcga catggagccg 300 gaggccggcg ctggcgccgc ggcctcttcg gctgcggacc cggccattcc tgaagaggta 360 tggaatatca agcaaatgat taagttgaca caggaacata tagaggccct attggacaaa 420 tttggtggag agcataaccc accatcaata tacctggagg cctatgaaga gtacaccag 480 aagctagatg cccttcagca aagagaacag cagcttttgg aatccctggt ttttcaaact 540 cccacagatg catcacggaa caaccccaag tcaccacaga aacctatcgt tagagtcttc 600 ctgcccaaca aacagaggac agtggtaccc gcaagatgtg gtgttacagt tcgagacagt 660 ctaaagaaag cactgatgat gagaggtctc atcccagaat gctgtgctgt ttacagaatt 720 caggatggag agaagaaacc aattggctgg gacacggaca tttcctggct tactggagag 780 gagttacatg ttgaagtact ggagaatgtc ccacttacaa cacacaactt tgtacggaaa 840 acttttttca ccttagcatt ttgtgacttt tgccgaaagc tgcttttcca gggtttccgt 900 tgtcaaacat gtggttataa atttcaccag cgttgtagta cagaggttcc actgatgtgt 960 gtaaattatg accaacttga tttgctgttt gtctccaagt tctttgagca tcacccagta 1020 ccacaggagg aggcctcctt cccagagact gcccttccat ctggatcctc ttccgcaccc 1080 ccctcagact ctactgggcc ccaaatcctc accagtccat ctccttcaaa atccattcca 1140 attccacagc ccttccgacc agcagatgaa gatcatcgca atcagtttgg gcaacgagac 1200 cggtcctcct cagctcccaa tgttcatata aacacaattg agcctgtgaa tatcgatgaa 1260 aaattcccag aagtggaatt acaggatcaa agggatttga ttagagacca ggggtttcgt 1320 ggtgatggag cccccttgaa ccaactgatg cgctgtcttc ggaaatacca atcccggact 1380 cccagccccc tcctccattc tgtccccagt gaaatagtgt ttgattttga gcctggccca 1440 gtgttcagag ggtcaaccac aggcttgtcc gccaccccgc ctgcctcatt acctggctca 1500 ctcactaacg tgaaagcctt acagaaatct ccaggtcctc agcgggaaag gaagtcatct 1560 tcttcctcat cctcggagga cagaagtcgg atgaaaacac ttggtagaag agattcaagt 1620 gatgactggg agattcctga tggacagatt acagtgggac agagaattgg atctgggtca 1680 tttggaactg tctacaaggg aaagtggcat ggtgatgtgg cagtgaaaat gttgaatgtg 1740 acagcaccca cacctcaaca gctacaggcc ttcaaaaatg aagtaggagt gctcaggaaa 1800 actcgacatg tgaatatcct ccttttcatg ggctattcta caaagccaca actggcaatt 1860 gttacacagt ggtgtgaggg ctccagctta tatcaccatc tccacatcat tgagaccaaa 1920 tttgagatga tcaaacttat agatattgct cggcagactg cacagggcat ggattactta 1980 cacgccaagt caatcatcca cagagacctc aagagtaata atatatttct tcatgaagac 2040 ctcacggtaa aaataggtga ctttggtcta gccacagtga aatctcggtg gagtgggtcc 2100 catcagtttg aacagttgtc tggatctatt ttgtggatgg caccagaagt aatcagaatg 2160 caagataaaa acccgtatag ctttcagtca gacgtgtatg cgtttgggat tgttctgtac 2220 gaactgatga ccggccagct accttattca aacatcaaca acagggatca gataattttt 2280 atggtgggac gaggatacct atctccagat ctcagtaagg tacggagtaa ctgtccaaaa 2340 gccatgaaga gattaatggc agagtgcctc aaaaagaaaa gagacgagag accactcttt 2400 ccccaaattc tcgcctccat tgagctgctg gcccgctcat tgccaaaaat tcaccgcagt 2460 gcatcagaac cttccttgaa tcgggctggt ttccaaacag aagattttag tctgtatgct 2520 tgtgcttctc cgaaaacacc catccaagca gggggatatg gagaatttgc agccttcaag 2580 tagccagtcc atcatggcag catctactct ttatttctta agtcttgtgt tcatacagtt 2640 tgttaacatc aaaacacagt tctgttcctc aaaaaatttt ttaaagatac aaaattttca 2700 atgcataagt tcatgtggaa cagaatggaa tttcctattc aacaaaagag ggaagaatgt 2760 tttaggaacc agaattctct gctgcccgtg tttcttcttc aacataacta tcacgtgcat 2820 acaagtctgc ccattcccaa gaagaaagag gagagaccct gaattctgcc cttttggtgg 2880 tcaggcatga tggaaagaat ttgctgctgc agcttgggaa aattgctatg gaaagtctgc 2940 cagtcgactt tgcccttcta accaccagat cagcctgtgg ctggtcatct gatggggcga 3000 tttccatcac caagcatcgt tcttgcctat tctgggatta tgttgtggag cactttccct 3060 gtccagcacc gttcatttct gagggatgga gtaaatgcag cattcccttg tgtagcgcct 3120 gttcagtcct cagcagctgc tgtcacagcg aagcttttta cagttaagtg gtgggggaga 3180 gttgaggaga gcctgcctcg gggcagagaa aagggggtgc tgcatcttct tcctcacctc 3240 cagctctctc acctcgggtt gccttgctca ctgggctccg cctaaccact caggctgctc 3300 agtgctggca cacattgcct tcttttctca ttgggtccag caattgagga gagggttggg 3360 ggattgtttc ctcctcaatg tagcaaattc tcaggaaaat acagtccata tcttcctctc 3420 agctcttcca gtcaccaaat acttacgtgg ctcctttgtc caggacataa aacaccgtgg 3480 acaacaccta attaaaagcc tacaaaactg cttactgaca gttttgaatg tgagacactt 3540 gtgtaattta aatgtaaggt acaggtttta atttctgagt ttcttctatt tttatttaaa 3600 agaagaaaat aattttcagt tttaattgga ataaatgagt acttcccaca agactatata 3660 ccctgaaaat tatatttttg ttaattgtaa acaactttta aagaataatt attatccttt 3720 tctctaccta aaaattatgg ggaatcttag cataatgaca attatttata ctttttaaat 3780 aaatggtact tgctggatcc acactaacat ctttgctaac aatcccattg tttcttccaa 3840 cttaactcct acactacatc ctacatcctc tttctagtct tttatctata atatgcaacc 3900 taaaataaac gtggtggcgt ctccattcat tctccctctt cctgttttcc ccaagcctgg 3960 tcttcaaaag gttgggtaat cggtccctga gctccctagc tggcaatgca actattaggg 4020 acattggagt tgcaggagag caggaagcct gtccccagct gttcttctag aaccctaaat 4080 cttatctttg cacagatcaa aagtatcacc tcgtcacagt tctccttagc ctttacttac 4140 aggtaatata aataaaaatc accatagtag taaagaaaac aactggatgg attgatgacc 4200 agtacctctc agagccagga atcttgaatc tccaggattt atacgtgcaa atttaaggag 4260 atgtacttag caacttcaag ccaagaactt ccaaaatact agcgaatcta aaataaaatg 4320 gaattttgag ttatttttaa agttcaaatt ataattgata ccactatgta tttaagccta 4380 ctcacagcaa gttagatgga ttttgctaaa ctcattgcca gactgtggtg gtggtggtgg 4440 tagtgtgcac ctttaatcca agcaactcag caatcagaat gaggtaaatc tctgtgaata 4500 caaggcctgc ctagtctgca gcgctagttc caggatagcc agggctacac acacaaaaac 4560 cctctctcaa aaaaaacaaa attaattagt tgataataaa aaataactaa agtatcatca 4620 aaggaaggcc tactggaagt tttatatatt cccagtaaat tgaaaaatat tctgaagtta 4680 ttaaccagtt agcaacaatg tgtttttaag tcttacataa acagagcaaa gtcttcaaat 4740 gtttagagc tgagaagata attgtgcttg atatgaaaaa tagcctctcc atatgatgtg 4800 ccacattgaa aggcgtcatt acccttttaa atacttctta atgtggcttt gttcccttta 4860 cccaggatta gctagaaaga gctaggtagg cttcggccac agttgcacat ttcgggcctg 4920 ctgaagaatg ggagctttga aggctggcct tggtggagga gcccctcagt gctggagggt 4980 ggggcgtgta cgcagcatgg aagtggtcta gacagagtgc aaagggacag acttctttct 5040 cattttagta tagggtgatg tctcacttga aatgagaaag tagagttgat attaaacgaa 5100 gctgtgccca gaaaccaggc tcagggtatt gtgagatttt ctttttaaat agagaatata 5160 aaagatagaa ataaatattt aaaccttcct tcttattttc tatcaaatag atttttttta 5220 tcatttgcaa acaacataaa aaaaggtttc ttttgtgggg ttttctttcc ttcttttttt 5280 tttttttttt tttttaagac tgcagataat cttgttgagc tcctcggaaa atacaaggaa 5340 gtccgtgttt gtgcagagcg ctttatgagt aactgtatag acagtgtggc tgcttcactc 5400 atcccagagg gctgcagctg tcggcccatg aagtggctgc agtgcctcgt gagatctgct 5460 ttgttttgtt tggagtgaag tctttgaaag gtttgagtgc aactatatag gactgttttt 5520 aaataagtag tattcctcat gaactttctc attgttaagc tacaggaccc aaactctacc 5580 actaagatat tattaacctc aaaatgtagt ttatagaagg aatttgcaaa tagaatatcc 5640 agttcgtact tatatgcatc ttcaacaaag attctctgtg acttgttgga tttggttcct 5700 gaacagccca tttctgtatt tgaggttagg agggcataat gaggcatcct aaaagacaat 5760 ctgatataaa ctgtatgcta gatgtatgct ggtaggggag aaagcattct gtaaagacat 5820 gatttaagac ttcagctctg tcaaccagaa accttgtaaa tacttcctgt cttggtgcag 5880 ccccgcccct ttgatcacac gatgttgtct tgtgcttgtc agacactgtc agagctgctg 5940 ttcgtccctc tgcagatctc acctgtcccc actgcacacc cacctcctgc ctcttgcaga 6000 cctcagcatc tagctttagt tggaaacagt tcagggttca ggtgacttct taaaaaaaaa 6060 aaaaaaccct acctcctcag aatgaggtaa tgaatagtta tttatttaaa gtatgaagag 6120 tcaggagcgc tcgaacatga aggtgattta agatggttcc tttcgtgtgt attgtagctg 6180 agcacttgtt tttgtcctaa agggcattat acatttaagc agtgattctg tttaaagatg 6240 tttttcttta aaggtgtagc tcagagtatc tgttgttgga attggtgcca gagtctgctt 6300 aatagatttc agaatcctaa gcttaagtca gtcgcatgaa gttaagtagt tatggtaaca 6360 ctttgctagc catgatataa ttctactttt taggagtagg tttggcaaaa ctgtatgcct 6420 tcaaagtgag ttggccacag ctttgtcaca tgcacagata ctcatctgaa gagactgccc 6480 agctaagagg gcggaaggat accctttttt cctacgattc gcttctttgt ccacgttggc 6540 attgttagta ctagtttatc agcaccttga ccagcagatg tcaaccaata agctattttt 6600 aaaaccatag ccagagatgg agaggtcact gtgagtagaa acagcaggac gcttacagga 6660 gtgaaatggt gtagggaggc tctagaaaaa tatcttgaca atttgccaaa tgatcttact 6720 gtgccttcat gatgcaataa aaaagctaac attttagcag aaatcagtga tttacgaaga 6780 gagtggccag tctggtttaa ctcagctggg ataatatttt tagagtgcaa tttagactgc 6840 gaagataaat gcactaaaga gtttatagcc aattcacatt tgaaaaataa gaaatggta 6900 aattttcagt gaaattatttt tttaaagcac ataatcccta gtgtagccag aaatatttac 6960 cacatagagc agctaggctg agatacagtc cagtgacatt tctagagaaa ccttttctac 7020 tcccacgggc tcctcaaagc atggaaattt tatacaaaat gtttgacatt ttaagatact 7080 gctgtagttt agttttgaaa tagtatgtgc tgagcagcaa tcatgtacta actcagagag 7140 agaaaacaac aacaaattgt gcatctgatt tgttttcaga gaaatgctgc caacttagat 7200 actgagttct cagagcttca agtgtaaact tgcctcccaa gtcctgtttg caaatgaagt 7260 tggctagtgc tactgactgc tccagcacat gatggaaggc agggggctgt ctctgaagtg 7320 tcttctataa agggacaata gaatagtgag agacctggtc agtgtgtgtc agctggacac 7380 tccatgctat gggacttgca tcttctgtcc tcaccatccc caagacattg tgctttcctc 7440 agttgtcctc tagctgtttc actcagacac caagatgaat tactgatgcc agaaggggcc 7500 aaaatggcca gtgtgttttg ggggttgtat cagttgactg gacaataact ttaatagttt 7560 cagatcattt atttttactt ccattttgac agacatttaa atggaaattt agtcctaact 7620 tttgtcattt gaaaggaaaa attaacagtt cctataagat acttttgagg tggaatctga 7680 catcctaatt ttttttcttt tcagtgggtt tgcagcgagg gtcttgtatg cactaggcaa 7740 gggttctacc actaagccac atttcccagg aaataaaatg ttaacagtta aaacatacac 7800 acaaatacac aaacacctta ttaccacttt agtaaagtga gagatgtgcg tcctttgtct 7860 cagtctccac gatttcagct gccccttgta tgaataactc agtctcgcta aactgtttac 7920 ttttatttac ctggtttgac tagttgcagc tatataacca gttgtgcatg aggacaacag 7980 ccagtgtgtt tgttttgttt ttggtttttt gtggtacatt ttttgtaaag aattctgtag 8040 attgaagtgc tctttgaaaa cagaactgag atatatttat tcttgttagc atcaaaaaac 8100 attttgtgca aatgatttgc ttttcctggc aggctgagta ccatatccag cgcccacaat 8160 tgcgggttcc catctaccat gtccacaggg gagacagacg ggaagcacat gaggggtgtg 8220 tttacagagt tgtaggagtt atgtagttct cttgttgcct tggaaatcac tgttgtttta 8280 agactgttga acccgtgtgt ttggctgggc tgtgagttac atgaagaaac tgcaaactag 8340 catatgcaga caaagctcac agactaggcg taaatggagg aaaatggacc aaaataaggc 8400 agggtgacac ataaaccttg ggcttcggag aaaactaagg gtggagatga actataatca 8460 cctgaataca atgtaagagt gcaataagtg tgcttattct aagctgtgaa cttcttttaa 8520 atcattcctt tctaatacat ttatgtatgt tccattgctg actaaaacca gctatgagaa 8580 catatgcctt tttattcatg ttaactacca gtttaagtgg ctaaccttaa tgtcttattt 8640 atcttcattt tgtattagtt tacataccag gtatgtgtgt gtgctgtact cttcttccct 8700 ttatttgaaa acacttttca ctgggtcatc tccttggcca ttccacaaca caactttggt 8760 ttggctttca atgtcacctt atttgatggc ctgtgtccca gtagcagaat ttatggtatt 8820 cccattgctg gctgctcttc cgaccctttg cttctacagc acttgtctct cctaagatag 8880 tcagaaacta actgatcagg ggatggactt caccattcat cgtgtctctt caattctatt 8940 aaatagacca ctcttgggct ttagaccagg aaaaaggaga cagctctagc catctaccaa 9000 gcctcaccct aaaaggtcac ccgtacttct tggtctgagg acaagtctcc actccagtaa 9060 gggagagggg aggaaatgct tcctgtttga aatgcagtga attcctatgg ctcctgtttc 9120 accacccgca cctatggcaa cccatataca ttcctcttgt ctgtaactgc caaaggttgg 9180 gtttatgtca cttcagttcc actcaagcat tgaaaaggtt ctcatggagt ctggggtgtg 9240 cccagtgaaa agatggggac tttttcatta tccacagacc tctctatacc tgctttgcaa 9300 aaattataat ggagtaacta tttttaaagc ttatttttca attcataaga aaaagacatt 9360 tattttcaat caaatggatg atgtctctta tcccttatcc ctcaatgttt gcttgaattt 9420 tgtttgttcc ctatacctac tccctaattc tttagttcct tcctgctcag gtcccttcat 9480 ttgtactttg gagtttttct catgtaaatt tgtataatgg aaaatattgt tcagtttgga 9540 tagaaagcat ggagaaataa ataaaaaaag atagctgaaa atcaaattga agaaatttat 9600 ttctgtgtaa agttatttaa aaactctgta ttatatttaa agaaaaaagc ccaacccccc 9660 aaaaagtgct atgtaattga tgtgaatatg cgaatactgc tataataaag attgactgca 9720 tggagaaa 9728 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BRAF_LCM_F <400> 5 tgcttgctct gataggaaaa tg 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BRAF_LCM_R <400> 6 agcctcaatt cttaccatcc ac 22 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LSL-BrafV600E Fwd <400> 7 cccaggctct ttatgagaa 19 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> LSL-BrafWT Rev <400> 8 agtcaatcat ccacagagac ct 22 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LSL-BrafV600E Rev <400> 9 gcttggctgg acgtaaacc 19

Claims (15)

A mutation in which the 600th valine is replaced with glutamic acid in the amino acid sequence of SEQ ID NO: 1, or a mutant in which the 637th valine is substituted with glutamic acid in the amino acid sequence of SEQ ID NO: 3; Or a nucleic acid molecule which encodes the BRAF mutant protein, wherein the composition is a composition for inducing brain metastasis by a brain somatic mutation of BRAF,
Wherein the BRAF mutant protein or a nucleic acid molecule encoding the BRAF mutant protein is expressed in neurons in the embryonic development stage to induce brain metastasis caused by a BRAF brain mutation caused by the formation of a brain somatic mutation of BRAF in a brain neuron ,
Composition for inducing brain metastasis by brain somatic mutation of BRAF. 2. The BRAF mutant protein of claim 1, wherein the amino acid sequence of the amino acid sequence of SEQ ID NO: 1 is substituted with glutamic acid at position 600, wherein the nucleotide sequence of SEQ ID NO: 2 is substituted with adenine at position 1799 Or encoded by a nucleotide sequence comprising a mutation, or
The BRAF mutant protein consisting of the amino acid sequence comprising a mutation in which the 637th valine is replaced with glutamic acid in the amino acid sequence of SEQ ID NO: 3 comprises a nucleotide sequence comprising a mutation in which the 1910th thymine is substituted with adenine in the nucleotide sequence of SEQ ID NO: Lt; / RTI &gt;
Composition for inducing brain metastasis by brain somatic mutation of BRAF. 2. The composition according to claim 1, wherein the brain metastasis is caused by activation of the mTOR signaling pathway, but is induced by brainstem mutation of BRAF. A nucleic acid molecule encoding a BRAF mutant protein consisting of a mutation in which the 600th valine in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamic acid or a mutant in which the 637th valine in the amino acid sequence of SEQ ID NO: 3 is substituted with glutamic acid; To produce a transgenic animal having a Cre-dependent condition BRAF mutation gene;
Mating said transgenic animal to produce an embryo-pregnant animal; And
And a step of injecting a vector containing a nucleic acid molecule encoding Cre recombinase at the time of the embryo into the lateral ventricle of the embryo to induce a Cre-dependent BRAF mutation. A method of producing an animal other than a human. 5. The method of claim 4, wherein said brain metastasis is not caused by activation of the mTOR signaling pathway. 5. The BRAF mutant protein according to claim 4, wherein the BRAF mutant protein consisting of the amino acid sequence comprising a mutation in which the 600th valine in glutamic acid is substituted in the amino acid sequence of SEQ ID NO: 1 has the nucleotide sequence of SEQ ID NO: 2 in which the 1799th thymine is substituted with adenine Or &lt; / RTI &gt; the nucleotide sequence comprising the &lt; RTI ID = 0.0 &gt;
The BRAF mutant protein consisting of the amino acid sequence comprising a mutation in which the 637th valine is replaced with glutamic acid in the amino acid sequence of SEQ ID NO: 3 comprises a nucleotide sequence comprising a mutation in which the 1910th thymine is substituted with adenine in the nucleotide sequence of SEQ ID NO: &Lt; / RTI &gt; [Claim 4] The animal according to claim 4, wherein the animal having cerebral apoplexy due to the BRAF brain somatic mutation exhibits at least one characteristic selected from the group consisting of nerve cell dendritic disorder and cortical abnormal laminae due to brain somatic mutation of BRAF &Lt; / RTI &gt; 5. The method according to claim 4, wherein the brain somatic mutation of BRAF is accompanied by the development of a neuronal cell tumor. A mutation in which the 600th valine is replaced with glutamic acid in the amino acid sequence of SEQ ID NO: 1, or a mutant in which the 637th valine is substituted with glutamic acid in the amino acid sequence of SEQ ID NO: 3; Or a nucleic acid molecule encoding said BRAF mutant protein,
Wherein the BRAF has at least one characteristic selected from the group consisting of a disorder of arrangement of dendrites of neurons due to a brain somatic mutation,
An animal other than a human having epilepsy caused by brain mutation of BRAF. 10. An animal according to claim 9, wherein the animal is a mammal or rodent other than a human, except for a human having brain growth caused by a brain somatic mutation of BRAF. 10. The animal according to claim 9, wherein the animal is a spontaneous seizure with epilepsy, except for a human having brain hemorrhage due to a brain somatic mutation of BRAF. 5. The method according to claim 4, wherein the vector is introduced into the lateral ventricle of the embryo during a period during which a cortical layer is formed in the embryo. 11. A method for treating brain metastasis of BRAF, comprising administering a candidate compound for treatment of brain metastasis to an animal other than a human having brain hematopoiesis caused by BRAF mutation according to any one of claims 9 to 11, Screening method for the treatment of cerebral infarction by brain mutation of BRAF. 14. The method of claim 13, wherein the brain stem treatment candidate material is administered by intracerebroventricular injection of the animal. 14. The method of claim 13, wherein the step of confirming whether or not the epileptic relief is performed is performed by measuring the electroencephalogram.

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