JPH11137257A - Neurralized protein, polynucleotide encoding the protein, and antibody recognizing the protein - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To identify a frequently deleted site in chromosome 10, identify a neuronal mutation-causing gene located in that site, and provide the gene. Further, the present invention provides a protein (neural mutation factor) encoded by the gene, and further provides an antibody against the protein. SOLUTION: The present invention provides a human neuronal mutation-causing gene which is present in a frequently deleted site in human chromosome 10.
The human neuronal mutation-causing gene has a high homology to the Drosophila neuralized gene and is a human homolog of the neuralized gene. Also provided is a neutralized gene in a mouse. The present invention also provides a protein encoded by the gene, and an antibody against the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a homologous protein of a Drosophila neuralized protein and a polynucleotide encoding the homologous protein.
In addition, the present invention relates to an antisense polynucleotide of the polynucleotide. The present invention also relates to an antibody that recognizes the homolog protein.

[0002]

BACKGROUND ART Among vertebrates, humans are organisms having the most highly differentiated nervous system. However, its analysis has been delayed due to its complex mechanism. In particular, the analysis of tumors occurring in the cerebral nervous system is delayed compared to other tumors. In the cranial nervous system, not only malignant tumors, but also benign tumors can be life-threatening for patients depending on the site of occurrence, and elucidation of the mechanism of the onset of cranial nerve tumors is awaited.

[0003] To date, it has been known that tumors in the cerebral nervous system, particularly abnormalities of malignant glioma, are derived from abnormalities of chromosome 10. However, the specific relationship between abnormalities such as deletion of chromosome 10 and clinical symptoms has not been clarified.

On the other hand, in Drosophila, many mutants have been obtained in the course of analysis of neurogenesis, and genetic analysis has been advanced. As a result, notch, delta, big brain, and neutralized (neu) have been used so far.
(raised), master mind (masterm)
ind), armondex,
Enhancer of Split
split) and the like are considered to be the causative genes of the mutation. Among the above genes, human homologs are obtained for Notch, Delta and Enhancer of Split.

[0005]

SUMMARY OF THE INVENTION The present invention is intended to clarify tumors and abnormalities of chromosome 10 in the cerebral nervous system.
It is an object of the present invention to identify a site having a high frequency of deletion in chromosome No. 1, identify a gene responsible for a neuronal mutation located in the site, and provide the gene. It is another object of the present invention to provide a protein (neural mutation factor) encoded by the gene, and to provide an antibody against the protein.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a human neuronal mutation-causing gene which is present at a frequently deleted site in human chromosome 10. Further, the present invention provides a mouse nerve mutation causing gene having homology with the human nerve mutation causing gene. These genes have a high homology to the Drosophila neuralized gene, and are a human homolog and a mouse homolog of the neuralized gene, respectively. According to the present invention, it has been clarified that the neutralized gene conventionally known in Drosophila is also present in vertebrates such as humans and mice, and more specifically in mammals.

[0007] The present invention also provides an antisense polynucleotide comprising a base sequence complementary to the above base sequence.

[0008] Further, the present application discloses a method for producing a neutralized protein. Specifically, a method is disclosed in which a transformant into which the neutralized cDNA has been introduced produces a neutralized protein. Also disclosed is a neutralized protein produced by the method. Further, a method for producing a mutant of a neutralized protein in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the neutralized protein, and a mutant of the neutralized protein produced by the method are provided. Disclosed.

[0009] The present invention also discloses an antisense polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of the polynucleotide encoding the neutralized protein or the polynucleotide encoding the mutant of the neutralized protein. is there. The antisense polynucleotide is included in the polynucleotide, but in the present specification, particularly when it is specified that the polynucleotide is a polynucleotide consisting of a base sequence of an antisense strand, it is referred to as an antisense polynucleotide. Representative antisense polynucleotides include antisense DNA and antisense RNA.

[0010] The present invention also discloses a polynucleotide which is a whole or a part consisting of 12 or more bases of a polynucleotide encoding a neutralized protein or a mutant of the neutralized protein. This polynucleotide can be used for producing a partial length protein of a neutralized protein or a mutant of a neutralized protein, respectively, for a part of the coding region. It can also be used as a probe.

[0011] The present invention also discloses an antisense polynucleotide that is a whole or a part of 12 or more bases of the antisense polynucleotide of the neutralized protein or the mutant of the neutralized protein. The antisense polynucleotide is capable of inhibiting the biosynthesis of a neutralized protein or a mutant of a neutralized protein, respectively. It can also be used as a probe.

The present invention also discloses a polynucleotide obtained by chemically modifying the above-mentioned polynucleotide (including an antisense polynucleotide).

[0013] The present invention also provides a method for detecting neutralized mRNA from human and mouse tissues and cell lines by analysis by Northern blot hybridization using a DNA probe. It discloses that a raised mRNA is expressed.

[0014] The present invention also discloses a homologous protein of a human neutralized protein encoded by the homologous gene of the human neuralized gene (also a homologous protein of the Drosophila neutralized protein). In addition, in this specification, when the origin animal name is not shown, it refers collectively to the neutralized protein in each vertebrate. The above neutralized protein can be produced by a transformant into which the neutralized gene has been introduced.

The present invention uses a polynucleotide encoding human neutralized, a polynucleotide encoding mouse neutralized or a part thereof consisting of 12 or more bases (part of the coding region) as a probe, The present invention also includes a method for obtaining a neutralized cDNA from a vertebrate, preferably a mammalian cDNA library, and a mammalian neutralized cDNA obtained by the method.

The probe includes a nucleotide sequence of a human neutralized cDNA and a mouse neutralized cDN.
A cDNA fragment consisting of a nucleotide sequence having a high homology with the nucleotide sequence of A or a polynucleotide obtained from the nucleotide sequence of the cDNA fragment (single-stranded DNA (including antisense strand), RNA for cDNA or (Including those chemically modified). Then, by hybridizing the probe with a cDNA library of another animal, it is possible to obtain a neutralized cDNA of the animal.

Further, the present invention relates to the above-mentioned cDNA (human neural cDNA and mouse neural cDNA).
DNA), which is a homologous protein of a human neutralized protein and a mouse neutralized protein.

The present invention also provides an antibody that recognizes the neutralized protein and a mutant of the neutralized protein. In the present application, it is disclosed that the antigenicity of the neutralized protein, that is, it is possible to generate an antibody from the neutralized protein.

The present invention also discloses the location on the chromosome of the human neutralized gene.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The homologous protein of the Drosophila neutralized protein of the present invention is not limited to the protein consisting of the amino acid sequence of SEQ ID NO: 1 or 3 in the sequence listing, but encodes these proteins. Using a polynucleotide or a part of the coding region thereof, in particular, a polynucleotide consisting of the base sequence of SEQ ID NO: 2 or 4 or a part of the base sequence of the coding region thereof as a probe, from a cDNA library of each animal. The protein encoded by the obtained cDNA is also included in the present invention.

The nucleotide sequence well conserved between the human neutralized gene and the mouse neutralized gene is, for example, the position 576 of the nucleotide sequence of the human neutralized cDNA described in SEQ ID NO: 2 in the sequence listing. 9 from G
It is the sequence up to C at position 38, and is 82% of the nucleotide sequence of mouse neutralized cDNA described in SEQ ID NO: 4 in the sequence listing.
It corresponds to the 444th place from the place. DN consisting of this base sequence
Using A as a probe, a full-length neuralized gene can be obtained from the cDNA library of the target animal by the following procedure.

1) The DNA to be used as a probe is Takara
Label using MEGA LABEL kit (registered trademark, manufactured by Takara Shuzo) according to the instruction manual. 2) Next, a DNA reaction solution having the following composition was prepared, and this solution was incubated at 37 ° C for 30 minutes, and then incubated at 70 ° C for 1 minute.
Heat treat for 0 min to inactivate enzyme. DNA probe (2 pmol / μl) 2 μl 10-fold phosphorylation buffer 2 μl [γ-32P] ATP (370 MBq / ml) (Amersham) 5 μl T4 polynucleotide kinase 1 μl Total 10 μl

3) TE50 (50 mM Tris-HC)
l pH 8.0, 1 mM EDTA)
ephadex G-25 (registered trademark, manufactured by Pharmacia) was packed in a 1.5 ml polyprep column (manufactured by Bio-Rad) so that the bed volume became 1 ml,
The DNA reaction solution subjected to the heat treatment in 2) is loaded on the column. 4) After that, 200 μl TE50 was flown through the column four times,
A labeled DNA probe is obtained from the fraction eluted with 200 μl of the second time.

5) A plaque cDNA probe in which 150 μl of the labeled DNA probe fraction obtained above was immobilized on a nitrocellulose membrane (the immobilization of a cDNA library on a nitrocellulose membrane was performed using Molecu
lar Cloning 2nd Edition (C
old Spring Harbor Laborat
ory Press, 1989) 9.38-9.40
This can be done according to the method described on the page. ) And the following conditions.

Prehybridization 6 × SSC 5 × Denhardt's 0.05% sodium pyrophosphate 100 μg / ml denatured herring sperm (denature
d herring sperm DNA) 0.5% SDS Total volume 50ml Reaction temperature 37 ° C Reaction time 1 hour

Hybridization 6 × SSC 1 × Denhardt's 0.05% sodium pyrophosphate 100 μg / ml denatured herring sperm (denature
d herring sperm DNA) 1 × 10 ⁶ cpm / ml cDNA probe Total volume 50 ml Reaction temperature 42 ° C. Reaction time 18 hours

6) The hybridized nitrocellulose membrane is washed once under the following conditions. 6 × SSC, 0.1% SDS 500 ml, temperature 40 ° C., time 20 minutes 3 × SSC, 0.1% SDS 500 ml, time 42 ° C., time 20 minutes

7) Apply the washed nitrocellulose membrane to an X-ray film (for example, Kodak XAR5 film).
Exposure overnight at 80 ° C. and take an autoradiograph. 8) From the obtained autoradiograph, determine the position of the positive plaque, and collect the corresponding plaque on agar in SM solution. 9) The collected plaque is again formed on an NZY agar medium by a conventional method, and fixed on a nitrocellulose membrane. 10) Repeat steps 5) to 9) three times to make a single positive plaque. Recover the plaque and add
Suspend in 1 SM solution to stabilize the phage. The cDNA isolated from the plaque is a neutralized cDN
A.

3. Large-scale preparation of neuralized cDNA 1) 50 μl of plaque phage suspended in SM solution and 20 μl of Y1090r-E. Coli were mixed at 37 ° C. for 15 minutes.
Leave for a minute. 2) Then, 10 μg / ml ampicillin containing 10 μg / ml
Transfer the solution mixed in 1) to ml NZY medium and incubate at 37 ° C for 6 hours. 3) Centrifuge at 8000 rpm for 5 minutes and collect the supernatant. 4) 1 ml of 5M NaCl and 1.1 g of polyethylene glycol 6000 are added to the supernatant and dissolved. 5) Place the solution on ice for 1 hour, then 10,000 rpm
centrifuge at 4 ° C. for 20 minutes. 6) Collect the precipitate and suspend in 700 μl of SM solution. 7) Add 500 μl of chloroform and stir to dissolve the remaining E. coli. 8) Centrifuge at 5000 rpm for 10 minutes to collect the aqueous layer.

9) 1 mg / ml RNase
A, 1 μl each of 5 mg / ml DNase I (both from Sigma) was added, and the mixture was allowed to stand at 37 ° C. for 1 hour.
Polyethylene glycol 6000 (0.8M NaC
l) is added to the mixture and left on ice for 30 minutes. 10) After centrifugation at 4 ° C. and 15000 rpm for 20 minutes, collect the precipitate. 11) 500 μl of SM solution, 50 μl of 5
M NaCl, 50 μl of 0.5 M EDTA was added,
Further, 400 μl of phenol is added and stirred to dissolve the phage and release the cDNA. 12) The solution is centrifuged at room temperature at 15000 rpm for 5 minutes, and the aqueous layer is collected. To this, 1 ml of ethanol is added, centrifuged at 15000 rpm for 20 minutes, and the liquid layer is discarded. 13) Wash the precipitate with 1 ml of 70% ethanol,
μl of TE solution (10 mM Tris-HCl (pH
8.0) Dissolve the precipitate in 1 mM EDTA)
Obtain a solution.

4. Determination of the base sequence of the neutralized cDNA The entire base sequence of the neutralized cDNA is determined by the die terminator method using an auto-synthesizer.

5. 3. Determination of amino acid sequence From the nucleotide sequence determined in step 2, the neutralized cDN
The amino acid sequence of A is determined.

6. 8. Preparation of transformant A transformant can be prepared by inserting the neutralized cDNA obtained above into an appropriate vector (eg, a TA cloning vector) and introducing it into a host (eg, Escherichia coli) in the same manner as described above.

[0034] Natural or artificial mutations (eg, Molecular Cloning 2nd E)
The method described in page 15.1-15.113) allows the polynucleotide to be altered without altering the main function of the polypeptide encoded by the polynucleotide. According to this method, also for the neutralized protein of the present invention, a protein consisting of an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence described in SEQ ID NO: 1 or 3 in the sequence listing, ie, a neutralized protein Can be produced. The homology between the neutralized protein and its mutant is preferably 75% or more and less than 100% at the amino acid level.

The amino acid sequence of the mutant of the neutralized protein of the present invention can be determined from the nucleotide sequence of the gene encoding the mutant. For example, it is possible to use a commercially available program (for example, Genetix-Mac (registered trademark, manufactured by Software Development Corporation)).

Due to the degeneracy of the genetic code, at least a part of the base sequence of the polynucleotide can be replaced with another type of base without changing the amino acid sequence of the polypeptide produced from the polynucleotide. . Therefore, the polynucleotide encoding the neutralized protein of the present invention includes all patterns of degeneracy.

The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing is a naturally-occurring human neutralized c.
The DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing is a naturally occurring mouse neutralized gene.

The present invention includes an antisense polynucleotide comprising the base sequence of the antisense strand of the coding region of the polynucleotide encoding the neutralized protein, and a derivative thereof. The antisense polynucleotide is capable of hybridizing to a polynucleotide encoding a neutralized polynucleotide, and if the hybridizing polynucleotide is a polynucleotide in a coding region, the polynucleotide encoded by the polynucleotide is used. It is possible to inhibit the biosynthesis of the peptide. The antisense polynucleotide for inhibiting polypeptide biosynthesis preferably comprises 15 or more bases. On the other hand, it is inappropriate to incorporate a full-length antisense polynucleotide into cells even if it is too long. When the antisense polynucleotide is incorporated into cells to inhibit the biosynthesis of the neutralized protein, 12 to 30 bases, preferably 15 to 30 bases are used.
Base to 25 bases, more preferably 18 bases to 2
It is preferable to use an antisense polynucleotide consisting of 2 or less bases.

The antisense polynucleotide of the present invention or a part thereof includes all nucleotides consisting of a plurality of nucleotides consisting of bases, phosphates and sugars, including non-naturally occurring nucleotides. The typical one is antisense D
NA and antisense RNA.

The antisense polynucleotide of the present invention can be prepared by subjecting the desired DN
Various antisense polynucleotide derivatives having high binding strength to A or mRNA, tissue selection, cell permeability, nuclease resistance and intracellular stability can be obtained.

From the viewpoint of ease of hybridization, it is generally considered to be desirable to design an antisense polynucleotide or a derivative thereof having a base sequence complementary to the base sequence of the region forming the stem loop. ing. The antisense polynucleotide of the present invention and a derivative thereof,
If necessary, a stem loop can be formed.

An antisense polynucleotide having a sequence complementary to the sequence near the translation initiation codon, ribosome binding site, capping site, or splice site can generally be expected to have a high expression suppressing effect. Therefore, the antisense polynucleotide of the present invention or a derivative thereof, including a complementary sequence of a translation initiation codon of a gene or mRNA encoding neuralized, a ribosome binding site, a capping site, a splice site, A high expression suppression effect is expected.

The derivatives generally known at present are preferably derivatives having at least one of enhanced nuclease resistance, tissue selectivity, cell permeability and binding strength.
Particularly preferred are derivatives having a phosphorothioate bond as a skeletal structure. The polynucleotide of the present invention and its derivatives also include derivatives having these functions or structures.

If the antisense polynucleotide is a natural type, it may be synthesized using a chemical synthesizer, or the antisense polynucleotide of the present invention may be prepared by a PCR method using a gene encoding a neutralized as a template. Can be. Some derivatives, such as a methylphosphonate type and a phosphorothioate type, can be chemically synthesized. In this case, the desired antisense polynucleotide can be obtained by performing the operation according to the instructions attached to the chemical synthesizer and purifying the obtained synthetic product by HPLC using reverse phase chromatography or the like. Alternatively, a derivative thereof can be obtained.

The polynucleotide encoding the neutralized polynucleotide of the present invention, its antisense polynucleotide or a part thereof (polynucleotide comprising a continuous base sequence of 12 or more bases)
It can be used as a probe for screening a neutralized gene from a cDNA library or the like. When the species of the target animal is the same as the species of the animal from which the polynucleotide as a probe is derived, even a non-coding region can be used. At this time, those having a GC content of 30 to 70% can be suitably used. Further, a polynucleotide consisting of a continuous base sequence of 15 or more bases is particularly preferred. The polynucleotide used as a probe may be a derivative. Usually, a sequence having the number of bases or more is recognized as a sequence having specificity. As a cDNA library to be used in screening using the probe, one prepared from mRNA can be preferably used. A group of cDNAs selected from these cDNA libraries by random sampling can be used as a sample to be searched. Also, commercially available products can be used.

For example, a DNA consisting of a continuous nucleotide sequence of 12 or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 2 or 4 in the sequence listing, a polynucleotide hybridizing to the DNA (antisense polynucleotide), c
It can be used as a probe for screening a neutralized gene from a DNA library or the like.

Further, Northern blot hybridization is carried out on mRNA derived from each tissue using a polynucleotide encoding the neutralized polynucleotide of the present invention, its antisense polynucleotide or a polynucleotide which is a part thereof as a probe. It is possible to find tissues in which mRNA derived from the neutralized gene is expressed.

When chemically synthesizing DNA or RNA,
It is well known to chemically modify side chains by methylating or biotinylating them, or by substituting O for S in the phosphate group. For example, when chemically synthesizing the DNA described in SEQ ID NO: 2 in the sequence listing, the chemical modification can be performed to synthesize a DNA different from the DNA itself shown in the sequence listing. Also, cDNA
Even a cDNA obtained from a library can be labeled with a radioisotope. Therefore, the DNA and RNA of the present invention can be obtained by using the above-mentioned chemically modified DN.
A, RNA or antisense polynucleotide is included in the scope. Chemically modified DNA or R
NA can exert both a protein-encoding function and a probe function, and a chemically-modified antisense polynucleotide has a function of inhibiting the biosynthesis of a probe or protein or a function of a probe. Can also be demonstrated.

It is possible to obtain a transformant by introducing the plasmid into an appropriate host such as Escherichia coli by a known method. It is possible to produce a neutralized protein or a mutant of the neutralized protein by culturing the transformant into which the neutralized gene of the present invention has been introduced and allowing the gene to be amplified or the protein to be expressed. Next, the product is collected and, if necessary, concentrated, solubilized, dialyzed, and various chromatographic operations are performed to purify the neutralized protein or a mutant of the neutralized protein of the present invention. It is possible.

There are various textbooks on culturing the transformant, and it is possible to produce a neutralized protein or a mutant of the neutralized protein based on the nucleotide sequence described in the present invention by a known method. It is. At this time, as the host, any of bacteria such as Escherichia coli, yeast, and animal cells can be used, and animal cells are particularly preferable. To introduce a gene into a cell, a ribosome method, an electroporation method, or the like can be used. In particular, it is preferable to use the nuclear microinjection method.

Purification methods for purifying the neutralized protein or the mutant of the neutralized protein from the obtained culture include immunoprecipitation, salting out, ultrafiltration, isoelectric point precipitation, and gel filtration. Method, electrophoresis, ion exchange chromatography, hydrophobic chromatography, antibody chromatography, etc., various affinity chromatography, chromatofocusing, adsorption chromatography, reverse phase chromatography, etc. Just do it.

In the production step, the neutralized protein or the mutant of the neutralized protein to be produced may be prepared as a fusion peptide with another polypeptide in a transformant. In this case, in the purification step, an operation of treating with a chemical substance such as bromocyan or an enzyme such as a prosthesis to cut out the neutralized protein or a mutant of the neutralized protein is required.

According to the present invention, the antigenicity of the neutralized protein is, as exemplified in Example 3, an oligopeptide comprising the neutralized protein obtained by the above method or an amino acid sequence specific to the neutralized protein. Is immunized to animals from which the neutralized protein is derived and animals other than humans, whereby antibodies can be easily obtained. Therefore,
The antibody that recognizes the neutralized protein of the present invention (hereinafter, sometimes referred to as a neutralized antibody) is used for immunizing an animal from which the neutralized protein is derived and an animal other than a human. An antibody obtained by Western blotting, ELISA, or the like, wherein the antibody recognizes the neutralized protein.
Antibodies confirmed by the SA method, immunostaining method (eg, tissue staining of frozen specimens or paraffin specimens) and the like are included in the range.

It is a commonly used method to use, as an immunogen, a protein obtained by binding a part of the protein to another carrier protein such as bovine serum albumin, even if it is a part of the protein. A part of the protein may be synthesized using, for example, a peptide synthesizer. In addition, as a part of protein, it is preferable that it is 8 or more amino acid residues. It is well known that, for a substance whose antigenicity has been revealed, if a polyclonal antibody can be obtained by immunization, a monoclonal antibody is produced by a hybridoma using the lymphocytes of the immunized animal ("" Antibodies A Laboratory
Manual "(Cold SpringHarbo)
r Laboratory Press, 1988) C
Chapter 6). Therefore, the neutralized antibody of the present invention includes a monoclonal antibody within its scope.

In the present invention, antibodies also include active fragments. An active fragment means a fragment of an antibody having antigen-antibody reaction activity,
Specifically, F (ab ') ₂ , Fab', Fab, Fv
And the like. For example, when the antibody of the present invention is digested with pepsin, F (ab ') ₂ is obtained, and when digested with papain, Fab is obtained. F (ab ') ₂ is 2-
Reduction with a reagent such as mercaptoethanol and alkylation with monoiodoacetic acid gives Fab '. Fv is a monovalent antibody active fragment in which a heavy chain variable region and a light chain variable region are linked by a linker. A chimeric antibody can be obtained by retaining these active fragments and substituting other portions with fragments of another animal.

The antibody of the present invention can be used for elucidating functions such as expression of neuralized and elucidating the formation mechanism of malignant tumor.

For the detection of neuralized, there are a method using an antibody and a method using an enzyme reaction.
As a method using an antibody, specifically, a method for detecting neutralized by using a labeled neutralized antibody, and detecting a neutralized by using a neutralized antibody and a labeled secondary antibody of the antibody Method. As the label, for example, a radioisotope (R
I), enzymes, avidin or biotin, or fluorescent substances (FITC, rhodamine, etc.) are used.

Examples of a method utilizing an enzyme reaction include a method for identifying an immunoreactive molecule using an ELISA method, an immunoagglutination method, and a Western blot method, or a method similar thereto.

[0059]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. Example 1 I. Identification of Chromosome 10 Deletion Position in Malignant Glioma (1) Preparation of Cells Forty-six human malignant glioma tissues and peripheral blood of malignant glioma patients were collected. Peripheral blood was sampled at 5 cc into an EDTA-added blood collection tube, and leukocyte nucleated cells were collected by specific gravity centrifugation using Ficoll Paque (Pharmacia). The tissue was treated with collagenase and trypsin on a sample of a 5 mm square piece or a biopsy sample removed by surgery to prepare a cell suspension.

(2) Preparation of DNA DNA was extracted from the obtained cell suspension using Qiagen Blood and Cell Culture DNA Kit (Qiagen) according to the attached protocol.

(3) Microsatellite analysis The obtained DNA was analyzed using primers dye-labeled at 16 microsatellite markers on the long arm of 10 chromosomes. As a result, in grade III and grade IV malignant gliomas, D10S among microsatellite markers on the long arm of chromosome 10
Loss of heterogeneity (LHO) in the gene containing 540 and D10S566 was frequently detected.

(4) STS Mapping Then, using a panel of whole genome radiation hybrids and a YAC clone, D10S566 was used.
Peripheral STS mapping was performed. As a result, D10S
566 exists in 10q25 and S around it
The positional relationship of TS markers was clarified.

II. Identification of neuralized genes EST database search Internet EST database (database in which sequences of mRNA fragments are registered, http: // w
ww. ncbi. nlm. nih. gov. / I ht
EST) at about 10q24 to 25 from the sequence (EST) registered in H.826.
34 were sorted out. The localization of this EST was further determined by PCR on the panel of the radiation hybrids and PCR of the YAC clone, and H82634 was found to be D10S540 and D10S566 on human chromosome 10.
It was found that the gene containing H82634 was a causative gene of malignant glioma.

2. Search for TIGR database Internet TIGR database (database in which sequences of mRNA fragments are registered, http: //
www. ncbi. nlm. nih. gov. / I h
tmml), a sequence having homology to H82634 was searched. As a result, H82635 becomes H
It was found to be the sequence of the complementary strand of 82634. The following base sequence A was determined from these two sequences. Nucleotide sequence A: CAGCTGGACG CCCCGNTGNA GGCCGTAGAC GTCCACCAGG GCCCAGAGCG GGTCGGCCGT GCGGACCCGC TGAAGAACAG CATAACAGCC GAGTCGTTGA TGCGGTGGAA GACACGGCCC TTCTTGTCCA CCCAGAATGC GATGATGTTG CCCTCATTGC CAAACTCCTC AGGCAGCGCT NTGGCCCAGA AGCCACTCTG GGACACCAGG TTCGGGGCAG GCGTACTTGG GCAGCGAGTC AGGGTGGATG CGGGACGGGT CCTTGCTGGT GAAGCCAGCC GCAGGCCCCG CTCCAGCACT GCTTCTTGGT GATCTTCAGC CTGACTTGCT CGTAGATGAG GACCGGGCGG TTGCTGAAGG TGATGNCGTT GCAGAAGCTG GCCTGCCTCT TGNACAGCCT TGTGGCTGAG GTCCATGAGG ATCTGGGAGC CCTTGGTGTG CGGGTGGAAG AGCAGCGGCG TGGCTGGGAG CCCCCGCTGG GCAGCACTGC CGGACAGTGC TTCTGCTTGT GGTGGCATCG GTGAGAAGTG ACGGGGAAGG GGCCCCCGAT AGAGTCGTGG AGAGTGCTCC GGGTGAT

3. Synthesis of primers From the human cDNA library,
The following primers were synthesized to obtain NA fragments. P1 primer 5'-TA as 5 'primer
GACGTCCA CCAGGGCCCA GAC-3 '(base sequence described in SEQ ID NO: 5 in the sequence listing), P2 primer as 3' primer
5′-CGGAGCACTC TCCACGACTC TAT-3 ′ (base sequence described in SEQ ID NO: 6 in the sequence listing) was designed and a DNA synthesizer (ABI
(Model 392). The synthesized primer was adjusted to 20 pmol / μl with distilled water (the synthesis of the subsequent primers was performed in the same manner).

4. PCR was performed under the following conditions using a human fetal brain cDNA library (manufactured by Clontech) as the PCR cDNA library. 0.8 μl of cDNA dNTPmix (2.5 mM each) 0.6 μl P1 primer 0.6 μl P2 primer 0.6 μl 3.3 × PCR buffer 4.5 μl 25 mM MgCl ₂ 0.72 μl Distilled water 6.68 μl Total 14.5 μl

A solution of the above composition was overlaid with 15 μl of mineral oil, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
1 minute "cycle was repeated 35 times. Finally, a fragment extension reaction was performed at 72 ° C. for 10 minutes to complete PCR.

After the reaction, the PCR product was subjected to mini-gel electrophoresis on a 1.5% agarose gel. About 0.6kb
A band considered to be a gene encoding a neutralized protein fragment was cut out and a PCR product was recovered. Further, the mini-gel electrophoresis was performed again on a part of the collected product, and it was confirmed that a band appeared at about 0.6 kb. Hereinafter, this DNA fragment is referred to as fragment A.

4. Integration into vector Fragment A was subcloned using pCRITA cloning vector kit (Invitrogen) under the following conditions. 5 μl of sterile distilled water 10 μl ligation buffer 1 μl pCRII vector 2 μl DNA fragment 1 μl T4 DNA ligase 1 μl Total 10 μl A reaction was performed overnight at 14 ° C. to obtain a ligation mixture.

5. Transformation Transformation was performed using a TA cloning kit. 3. Add 50 μl of E. coli to 2 μl of 0.5 M 2-mercaptoethanol on ice and Was added and left for 30 minutes, followed by incubation in a water bath at 42 ° C. with shaking at 225 rpm. Next, the Escherichia coli was spread on an LB agar plate to which ampicillin, X-Gal and IPTG were added.
After incubation at 37 ° C. for 18 hours, white and blue colonies appeared.

6. Mini culture Four white colonies were selected from the above-mentioned plate, placed in a tube containing 3 ml of LB medium (ampicillin added) per colony, and cultured with shaking at 37 ° C. overnight.

7. Mini-preparation Alkali method (for example, Molecular Cl
oning 2ndEdition 1.25-1.2
The plasmid was prepared by the method described on page 8).

8. DNA sequence 7. Take 1 μl of the plasmid prepared in
Dilute with 99 μl of TE. The absorbance at 260 nm (A260) was measured and the DNA value was calculated (A260
Was calculated with the value of 1.0 being 50 μg / ml). A26
The plasmid was diluted with TE so that the DNA became 1 μg / ml from the 0 value. The base sequence of fragment A was determined by the dye terminator method using an auto-synthesizer model 373S manufactured by ABI.

9. 5 'extension reaction From the base sequence of fragment A, P3 primer 5'-AGCAC
TGCCG GACAGTGCTT CTG-3 ′ (base sequence described in SEQ ID NO: 7 in the sequence listing) was synthesized. Using this primer, a single P
CR was performed under the following conditions. cDNA 1 μl dNTPmix 0.6 μl P3 primer 1 μl 3.3 × PCR buffer 4.5 μl 25 mM MgCl ₂ 0.72 μl distilled water 6.68 μl total 14.5 μl

15 μl of mineral oil was overlaid on the liquid having the above composition, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1.5 minutes "was repeated 50 times. Subsequently, a fragment extension reaction was performed at 55 ° C. for 2 minutes and finally at 72 ° C. for 10 minutes to complete PCR.

Using this PCR product as a template, a second PC
R was performed. P4 primer 5'-GTGGTGGCAT CGGTGAGA
AG TGA-3 ′ (base sequence described in SEQ ID NO: 8 in the sequence listing) was synthesized. Using this primer and the T3 primer 5′-ATTAACCCTC ACTAAAG-3 ′ (base sequence described in SEQ ID NO: 9 in the sequence listing) as a 5 ′ primer under the following conditions:
CR was performed. PCR product 1 μl dNTPmix 1 μl P4 primer 1 μl T3 primer 1 μl 3.3 × PCR buffer 7.5 μl 25 mM MgCl ₂ 1.2 μl distilled water 11.8 μl Total 24.5 μl

20 μl of mineral oil was overlaid on the liquid having the above composition, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1 minute "was repeated 40 times. Subsequently, a fragment extension reaction was performed at 55 ° C. for 2 minutes and finally at 72 ° C. for 10 minutes to complete PCR. This DNA fragment is hereinafter referred to as fragment B.

10. 3 'extension reaction From the nucleotide sequence of fragment A, P5 primer 5'-GGCCG
TGTCT TCCACCGCAT CAA-3 ′ (base sequence described in SEQ ID NO: 10 in the sequence listing) was synthesized. Using this primer,
Single PCR was performed using the human fetal brain cDNA library as a template under the following conditions. cDNA 1 μl dNTPmix 0.6 μl P5 primer 1 μl 3.3 × PCR buffer 4.5 μl 25 mM MgCl ₂ 0.72 μl Distilled water 6.68 μl Total 14.5 μl

15 μl of mineral oil was overlaid on the liquid having the above composition, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1.5 minutes "was repeated 50 times. Finally, a fragment extension reaction was performed at 72 ° C. for 10 minutes to perform PC
R completed.

Using this PCR product as a template, a second PC
R was performed. P6 primer 5'-CTCGGCTGTT ATGCTGTT
CT TCA-3 '(base sequence described in SEQ ID NO: 11 in Sequence Listing)
Was synthesized. Using this primer and T7 primer 5′-AATACGACTC ACTATAG-3 ′ (base sequence described in SEQ ID NO: 12 in the sequence listing) as a 5 ′ primer, PCR was performed under the following conditions. PCR product 1 μl dNTPmix 1 μl P6 primer 1 μl T7 primer 1 μl 3.3 × PCR buffer 7.5 μl 25 mM MgCl ₂ 1.2 μl Distilled water 11.8 μl Total 24.5 μl

20 μl of mineral oil was overlaid on the liquid having the above composition, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1 minute "was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C. for 10 minutes to complete PCR. The DNA fragment thus obtained is hereinafter referred to as fragment C.

11. DNA sequencing Fragment B and fragment C were subjected to direct sequencing by the dye terminator method based on the P4 primer or the P6 primer.

12. 3 'extension reaction Based on the base sequence of fragment C, P7 primer 5'
-GTGGACGCCT CGCAGCCGCT TTG-3 '(SEQ ID NO: 1 in the sequence listing)
3) and P8 primer 5'-CGATGAGTGC
ACCATTTGCT ATG-3 ′ (base sequence described in SEQ ID NO: 14 in the sequence listing) was synthesized. First, single PCR was performed using the human fetal brain cDNA library as a template with the P7 primer under the following conditions. cDNA 1 μl dNTPmix 0.6 μl P7 primer 1 μl 3.3 × PCR buffer 4.5 μl 25 mM MgCl ₂ 0.72 μl Distilled water 6.68 μl Total 14.5 μl

15 μl of mineral oil was overlaid on the liquid having the above composition, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1.5 minutes "was repeated 50 times. Finally, a fragment extension reaction was performed at 72 ° C. for 10 minutes to perform PC
R completed.

A second PC using this PCR product as a template
R was performed. PCR was performed using the P8 and T3 primers under the following conditions. PCR product 1 μl dNTPmix 1 μl P8 primer 1 μl T3 primer 1 μl 3.3 × PCR buffer 7.5 μl 25 mM MgCl ₂ 1.2 μl Distilled water 11.8 μl Total 24.5 μl

20 μl of mineral oil was overlaid on the liquid having the above composition, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1 minute "was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C. for 10 minutes to complete PCR. The DNA fragment thus obtained is hereinafter referred to as fragment D.

13. DNA sequence Fragment D was directly sequenced by the dye terminator method based on the P8 primer.

14. Based on the nucleotide sequences of fragments A, B, C and D,
Sense primer 5'-AGAGCAGCAG AGGTGGCTGC ACT-3 '
(Base sequence described in SEQ ID NO: 15 in the sequence listing) was replaced with an antisense primer 5′-GGCTTGTTCC TCAGCTGGGA CTG-3 ′
(Base sequence described in SEQ ID NO: 16 in the sequence listing),
Using this, PCR was performed using the human fetal brain cDNA library as a template under the following conditions. cDNA 1 μl dNTPmix 1 μl Sense primer 1 μl Antisense primer 1 μl 3.3 × PCR buffer 7.5 μl 25 mM MgCl ₂ 1.2 μl Distilled water 11.8 μl Total 24.5 μl

15 μl of mineral oil was overlaid on the liquid having the above composition, left at 96 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1.5 minutes "was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C. for 10 minutes to perform PC
R completed. This full-length cDNA was sequenced by the dye terminator method. It was confirmed that the desired cDNA containing the full-length coding region was obtained, and this gene was identified as a human homolog of the Drosophila neutralized gene. This base sequence is shown as SEQ ID NO: 2 in the sequence listing. The amino acid sequence of the human neutralized protein encoded by this gene is shown in SEQ ID NO: 1 in the sequence listing.
Shown in

15. Preparation of Transformant Full-length Human Neuralized cDNA Obtained Above
Is used for the EcoRI- plasmid vector pBTM116.
BamHI-KpnI of pBTM116HA with 83 mer linker containing HA tag inserted at PstI site
Inserted into the site. Escherichia coli D
Transformants were prepared by introduction into H5α.

Example 2 Expression of neutralized mRNA in each tissue 2 μg of each of poly A + RNA (mRNA) of human tissues and poly A + RNA (mRNA) of various human cells was blotted on a membrane (Human).
Multiple Tissue Northern
Blot I, II and Human Cance
r Cell Line Multiple Tissue
ue Northern Blot (manufactured by Clonetech)) and fragment A labeled with ³² P-CTP using a random prime labeling kit (registered trademark, manufactured by Takara Shuzo) under the conditions of high stringency and Molecular Cloning. A Lab
Oratory Manual Second Edi
The hybridization was carried out according to the description on pages 7.39 to 7.52, and analysis was performed by Northern blot hybridization.

As a result, it was found that the neutralized mRNA was expressed in brain and muscle tissues.

As a result, no clear expression was observed for each cell.

From the above results, it is considered that the neuralized gene and the neuralized protein are nerve-specific molecules.

Example 3 Preparation of Antibody Recognizing Neurralized Protein Preparation of Antigen The following peptide A and peptide B were synthesized. Peptide A: 1 from Leu at position 123 in the amino acid sequence of the neutralized protein shown in SEQ ID NO: 1 in the sequence listing
A peptide consisting of 21 amino acids up to the 43rd Cys. Peptide B: 2 from Pro at position 272 of the amino acid sequence of the neutralized protein described in SEQ ID NO: 1 in the sequence listing
A peptide consisting of amino acids obtained by adding cysteine (Cys) to the N-terminal of 20 amino acids up to the 91st Asp. Cysteine was added to bind to keyhole limpet hemocyanin (KHL). The synthesis was outsourced to Iwaki Glass Co., Ltd. Maleimidated KLH of 2 mg of synthesized peptide
(Pierce) 2 mg. The reaction was performed according to the method described in the instructions of the Pierce kit.

2. Immunization For antigens (Peptide A and Peptide B), 100 μl of antigen solution (1 μg / ml), 0.5 ml of PBS and 0.5 ml of Freund's complete adjuvant (manufactured by Difco) were taken in a syringe and mixed to form an emulsion. Was inoculated subcutaneously in four places on the back. 1
A week later, a second immunization was performed. From the second time, immunization was performed by changing the adjuvant to Freund's complete adjuvant (manufactured by Difco). Other operations are the same as in the first operation. After the second time, immunization was performed 6 times in total at one week intervals.

3. Antibody Purification One week after the final immunization, blood was collected. This blood was allowed to stand at room temperature for 3 hours, and after sufficient blood coagulation, 3,000 rpm
After centrifugation at 5 m for 5 minutes, the supernatant (serum) was collected.
Saturated ammonium sulfate was added to the serum to a final concentration of 50% and salted out. This sample was centrifuged to precipitate a fraction containing the antibody. Thereafter, the precipitate was dissolved in PBS, and further subjected to salting-out for PBS. Thereafter, the antibody was affinity-purified using a Protein G Sepharose column (registered trademark, manufactured by Pharmacia). As a result, a total of 370 mg of an IgG fraction was obtained.

The IgG fraction was affinity-purified using a column prepared by binding the peptide used for immunization to NHS-activated Sepharose (Pharmacia) according to the attached manual. as a result,
A total of 5 mg of purified antibody was obtained.

4. Measurement of antibody titer The titer of the obtained purified antibody was measured by ELISA. (1) Antigen solution (peptide A and peptide B) was diluted to 25 μg / ml with PBS, respectively, and the solution was diluted to 96 wells.
ISA plate (Xenobind)
50 μl was dispensed into each well of a registered trademark (manufactured by Xenopore) and left at 4 ° C. overnight. (2) Discard the antigen solution and add Block Ace (registered trademark, manufactured by Dainippon Pharmaceutical Co., Ltd.) diluted 4 times with distilled water to each well for 20 times.
Blocking was performed by dispensing 0 μl each and leaving at room temperature for 2 hours.

(3) The blocking solution was discarded, and a purified antibody was added as a primary antibody. Purified antibody is available in 96 wells
10 μg / ml, 5 μg / ml, 2.5
μg / ml,.
50 μl was dispensed into each well until the column. The antibody concentration in the twelfth row is about 0.5 μg / ml. As a control, IgG purified from a non-immunized rabbit was used. After dispensing each antibody, the reaction was carried out at room temperature for 1 hour. (4) Discard the antibody solution, and add 0.05% Tween20 / PB.
Wash the plate four times with S, then use P as a secondary antibody solution.
50 μl was dispensed into each well of a biotinylated rabbit IgG antibody (manufactured by Vector) diluted 1000-fold with BS. Thereafter, the reaction was performed at room temperature for 30 minutes. (5) After discarding the secondary antibody, 0.05% Tween 20
The plate was washed four times with / PBS, and an ABC solution (manufactured by Vector) diluted 1000 times was dispensed into each well in an amount of 50 μl. Then, it was left at room temperature for 30 minutes. (6) After discarding the ABC solution, 0.05% Tween 20
Wash the plate 4 times with / PBS and add orthophenylenediamine (OPD) -H ₂ O ₂ / PBS to each well
Dispensed in μl. After the color was sufficiently developed, the reaction was stopped with 2N sulfuric acid, and the absorbance at 490 nm was measured with a microplate reader (manufactured by Bio-Rad).

As a result, both antibodies obtained by immunizing peptide A (hereinafter sometimes referred to as antibody A) and antibodies obtained by immunizing peptide B (hereinafter sometimes referred to as antibody B) Was higher than the control.

Example 4 Western Blot (1) EcoRI of plasmid vector pBTM116
-BamHI-Kpn of pBTM116HA with 83 mer linker containing HA tag inserted at PstI site
The coding region of human neutralized cDNA was inserted into the I site. This plasmid was introduced into COS cells. (2) COS into which the neuralized cDNA has been introduced
The cells and their parent strains (cells into which the neuralized gene was not introduced), human brain tissue and muscle tissue were collected and lysed. Cells are 1 × 10
^Six brain tissues were human brain protein medley (Human Brain pr
protein Medley) was used. Cell lysate
68 mM Tris-HCl, 14% glycerol, 3
% SDS, 0.1 M DTT, 10 μg / ml soybean trypsin inhibitor, 1 μg / ml aprotinin, 1 m
M phenylmethylsulfonyl fluoride (PMS
F) 1 μg / ml leupeptin was used. 100 μl of the cell lysate was added to the cell pellet, and the mixture was stirred well to dissolve. After boiling for 10 minutes, centrifugation was performed at 15000 rpm for 10 minutes, and the supernatant was collected.

(3) 10-20 per 10 μl of supernatant
Electrophoresis on a% gradient gel. (4) After electrophoresis, the DNA was transferred to a nitrocellulose membrane using a trans-blot system (manufactured by Marisol). (5) Nitrocellulose membrane is 10% skim milk / PB
S, immersed in 0.1% Tween20, left for 1 hour, and blocked. Then, 0.3% Tween20 / P
Washed twice with BS for 5 minutes each. (6) The anti-neuralized antibody prepared in Example 3 was
After dilution with PBS to μg / ml, the mixture was added to a nitrocellulose membrane and reacted at room temperature for 40 minutes. Then 0.3%
The wells were washed three times for 5 minutes each with Tween 20 / PBS. (7) Next, peroxidase-labeled anti-rabbit IgG
A solution obtained by diluting (Amersham) 20,000-fold with PBS was added to the nitrocellulose membrane, and further reacted at room temperature for 40 minutes. Then, 0.3% Tween20 / PB
Washed three times with S for 5 minutes each.

(8) Next, 5 ml of the color developing solution of the ECL kit (manufactured by Amersham) was added to the nitrocellulose membrane and reacted for 1 minute. Thereafter, the membrane was exposed to an X-ray film for 20 seconds, developed, and photographed. As a result, when any of the antibodies was used, a band of the neutralized protein was detected in each of the lysates of the COS cells, brain tissue, and muscle tissue into which the neutralized gene was introduced. Thus, it was confirmed that the antibodies A and B recognized the neutralized protein.

Example 5 Tissue Staining Immunohistostaining was performed on human nervous system tissue and human brain tissue using Antibody A and Antibody B by the following procedure. (1) Human brain tissue was placed in a compound to prepare a frozen block. The block was cryostat-sectioned on a cryostat, and the section was mounted on a slide glass. (2) Next, the frozen section tissue was fixed by performing 10% buffered formalin treatment for 10 minutes. Then, PBS
(PH 7.5) and washed three times for 5 minutes each. Furthermore, 0.
The cells were treated with 3% H ₂ O ₂ / methanol for 30 minutes and washed twice with PBS. (3) 0.15% donkey normal serum / PBS was placed on a tissue on a slide glass and left at room temperature for 1 hour to perform blocking.

(4) Antibody A and antibody B at 1 μg / ml
Were diluted with PBS and added to the tissue on the slide glass, and reacted at room temperature for 1 hour. After that, PB
The plate was washed with S (pH 7.5) three times for 5 minutes each. (5) Biotinylated rabbit I diluted 1000 times with PBS
The gG antibody (Amersham) was added to the tissue and reacted at room temperature for 1 hour. Thereafter, the plate was washed with PBS (pH 7.5) three times for 5 minutes each. Peroxidase labeled avidin-
After adding a biotin complex solution and reacting for 40 minutes, P
Washed three times for 5 minutes each with BS. (6) 0.1% diaminobenzidine (DAB), 0.0
The slide glass was immersed in a 2% H ₂ O _{2 /} PBS (pH 7.5) solution and reacted at room temperature for 10 minutes. Thereafter, the slide glass was transferred to distilled water to stop the reaction. After hematoxylin staining, the cells were washed with running water. Regardless of which antibody was used, it was observed that the neuralized protein was stained in the brain tissue.

Example 6 Mouse neutralized cDN
Isolation of A and determination of its nucleotide sequence and amino acid sequence encoded by the gene Acquisition of DNA Fragment A mouse neonatal brain cDNA library (Clontech) was used as a cDNA library, and PCR was performed under the following conditions. Single PCR was performed from the nucleotide sequence of fragment A using the P3 primer and the mouse neonatal brain cDNA library as a template under the following conditions. Mouse neonatal brain cDNA library (1 μg / ml) 1 μl dNTPmix 0.6 μl P3 primer 1 μl 3.3 × PCR buffer 4.5 μl 25 mM MgCl ₂ 0.72 μl distilled water 6.68 μl total 14.5 μl

15 μl of mineral oil was overlaid on the liquid having the above composition, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1.5 minutes "was repeated 50 times. Subsequently, a fragment extension reaction was performed at 55 ° C. for 2 minutes and finally at 72 ° C. for 10 minutes to complete PCR.

A second PC using the PCR product as a template
R was performed. PCR was performed using the P4 primer and the T3 primer under the following conditions. PCR product 2 μl dNTPmix 1 μl P4 primer 1 μl T3 primer 1 μl 3.3 × PCR buffer 7.5 μl 25 mM MgCl ₂ 1.2 μl Distilled water 11.8 μl Total 24.5 μl

20 μl of mineral oil was overlaid on the liquid having the above composition, left at 94 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1 minute "was repeated 40 times. Subsequently, a fragment extension reaction was performed at 55 ° C. for 2 minutes and finally at 72 ° C. for 10 minutes to complete PCR. This DNA fragment is hereinafter referred to as fragment MA.

The fragment MA obtained by the 5 ′ extension reaction obtained above was subjected to minigel electrophoresis (0.75% agarose gel), and the fragment MA band was cut out from the gel. Fragment MA was collected using GeneClean (manufactured by Bio-101), and bands were checked by minigel electrophoresis.

Take 1 μl of the fragment MA and 99 μl
Was diluted with TE. Absorbance at 260 nm (A26
0) was measured and the DNA concentration was calculated (A260 was 1.
The DNA concentration at 0 was 50 μl / ml. ). D
The fragment MA was diluted with TE so that the NA concentration was 1 μl / μl.

[0113] The diluted solution was subjected to DNA sequencing using an auto-sequencer (ABI model 373A) by a dye terminator method using a T3 primer, and the base sequence of the fragment MA was determined.

[0114] 2. Acquisition of mouse neutralized cDNA Sense primer P9 5′-GACTCCATCG GGGGCTCCTT C corresponding to a part of fragment MA as 5 ′ primer
CC-3 '(base sequence described in SEQ ID NO: 17 in the sequence listing)
Antisense primer P10 as 3 'primer
5′-CTAGGAGCTGCGGTAGGTCT TGA-3 ′ (base sequence described in SEQ ID NO: 18 in the sequence listing) was synthesized using a DNA synthesizer (ABI model 392).

A PCR was carried out under the following conditions using a mouse neonatal brain cDNA library (Clontech) as a template and the above primers. cDNA 1 μl dNTPmix 1 μl Sense primer 1 μl Antisense primer 1 μl 3.3 × PCR buffer 7.5 μl 25 mM MgCl ₂ 1.2 μl Distilled water 11.8 μl Total 24.5 μl

15 μl of mineral oil was overlaid on the liquid having the above composition, left at 96 ° C. for 5 minutes, and 0.5 μl of Taq polymerase XL (registered trademark, manufactured by Takara Shuzo) was added.
"94 ° C. for 30 seconds, 60 ° C. for 30 seconds, followed by 72 ° C.
For 1.5 minutes "was repeated 40 times. Finally, a fragment extension reaction was performed at 72 ° C. for 10 minutes to perform PC
R completed.

The full-length cDNA was sequenced by the dye terminator method. This gene was identified as a mouse homologue of the Drosophila neuralized gene. This base sequence is shown as SEQ ID NO: 4 in the sequence listing. The amino acid sequence of the mouse neutralized protein encoded by this gene is shown in SEQ ID NO: 3 in the sequence listing.

3. Preparation of Transformant The mouse neutralized gene having the above nucleotide sequence was inserted into a TA cloning site of a pCRII vector (registered trademark, manufactured by Invitrogen), and the vector was introduced into Escherichia coli DH5α to prepare a transformant. .

[0119]

EFFECTS OF THE INVENTION The Drosophila neutralized gene is one of a group of genes that operate during the process of neuronal cell formation, and its deletion causes abnormal proliferation of immature neurons during the larval stage in Drosophila. It is considered to be a gene that controls proliferation / differentiation induction signals of lineage cells. According to the present invention, it has been clarified that the neutralized gene conventionally known in Drosophila is also present in vertebrates such as humans and mice, and more specifically in mammals. The human neutralized gene is highly expressed in brain and muscle tissues and is a promising candidate as one of the genes involved in malignant tumor formation. The neutralized protein of the present invention, the gene encoding the protein, the antisense gene of the gene, and the antibody recognizing the protein are effective tools as reagents for analyzing brain tumors.

[0120]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 574 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Gly Asn Asn Phe Ser Ser Ile Pro Ser Leu Pro Arg Gly Asn Pro 1 5 10 15 Ser Arg Ala Pro Arg Gly His Pro Gln Asn Leu Lys Asp Ser Ile Gly 20 25 30 Gly Pro Phe Pro Val Thr Ser His Arg Cys His His Lys Gln Lys His 35 40 45 Cys Pro Ala Val Leu Pro Ser Gly Gly Leu Pro Ala Thr Pro Leu Leu 50 55 60 Phe His Pro His Thr Lys Gly Ser Gln Ile Leu Met Asp Leu Ser His 65 70 75 80 Lys Ala Val Lys Arg Gln Ala Ser Phe Cys Asn Ala Ile Thr Phe Ser 85 90 95 Asn Arg Pro Val Leu Ile Tyr Glu Gln Val Arg Leu Lys Ile Thr Lys 100 105 110 Lys Gln Cys Cys Trp Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys 115 120 125 Asp Pro Ser Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro 130 135 140 Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu 145 150 155 160 Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys Gly 165 170 175 Arg Val Phe His Arg Ile Asn Asp Ser Ala Val Met Leu Phe Phe Ser 180 185 190 Gly Val Arg Thr Ala Asp Pro Leu Trp Ala Leu Val Asp Val Tyr Gly 195 200 205 Leu Thr Arg Gly Val Gln Leu Leu Asp Ser Glu Leu Val Leu Pro Asp 210 215 220 Cys Leu Arg Pro Arg Ser Phe Thr Ala Leu Arg Arg Pro Ser Leu Arg 225 230 235 240 Arg Glu Ala Asp Asp Ala Arg Leu Ser Val Ser Leu Cys Asp Leu Asn 245 250 255 Val Pro Gly Ala Asp Gly Asp Glu Ala Ala Pro Ala Ala Gly Cys Pro 260 265 270 Ile Pro Gln Asn Ser Leu Asn Ser Gln His Ser Arg Ala Leu Pro Ala 275 280 285 Gln Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg Ala Gly Ala His 290 295 300 Val Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Val Glu His Gly Arg 305 310 315 320 Asp Glu Arg Ala Leu Val Phe Thr Ser Arg Pro Val Arg Val Ala Glu 325 330 335 Thr Ile Phe Val Lys Val Thr Arg Ser Gly Gly Ala Arg Pro Gly Ala 340 345 350 Leu Ser Phe Gly Val Thr Thr Cys Asp Pro Gly Thr Leu Arg Pro Ala 355 360 365 Asp Leu Pro Phe Ser Pro Glu Ala Leu Val Asp Arg Lys Glu Phe Trp 370 375 380 Ala Val Cys Arg Val Pro Gly Pro Leu HisSer Gly Asp Ile Leu Gly 385 390 395 400 Leu Val Val Asn Ala Asp Gly Glu Leu His Leu Ser His Asn Gly Ala 405 410 415 Ala Ala Gly Met Gln Leu Cys Val Asp Ala Ser Gln Pro Leu Trp Met 420 425 430 Leu Phe Gly Leu His Gly Thr Ile Thr Gln Ile Arg Ile Leu Gly Ser 435 440 445 Thr Ile Leu Ala Glu Arg Gly Ile Pro Ser Leu Pro Cys Ser Pro Ala 450 455 460 Ser Thr Pro Thr Ser Pro Ser Ala Leu Gly Ser Arg Leu Ser Asp Pro 465 470 475 480 Leu Leu Ser Thr Cys Ser Ser Gly Pro Leu Gly Ser Ser Ala Gly Gly 485 490 495 Thr Ala Pro Asn Ser Pro Val Ser Leu Pro Glu Ser Pro Val Thr Pro 500 505 510 Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys Tyr Glu His Ala 515 520 525 Val Asp Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys Tyr Ala 530 535 540 Cys Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys Cys Pro Ile Cys 545 550 550 555 560 Arg Arg Pro Ile Lys Asp Ile Ile Lys Thr Tyr Arg Ser Ser 565 570

SEQ ID NO: 2 Sequence length: 2207 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence CCGAACGCCC ACGCCAGCGA CCCTGACTCT ATGGCCCCGG GGGAGCGCGC CGGAGCCGCC 60 GCGCCGCCCA CCCCCAGCCG GAACCCGTAGGCGTCC AGCCCCGGGC 120 GTCCCCGGCC CCGGCCCAGG GCCCTGCTTG TGGCCCCCGC TCCCGCCACG GGGCATGGGA 180 GGCAGGTAGC CCAGCTCGCG CCAGGACACC CGTGGCGGGC GGAACCCGCC AAGGACCGCG 240 AAGTCCAGAG AAAGGAAGCT GAGGAGCTGC CCGCCCGCCC CCGGCTGCAG CCCCAGCAGG 300 GCCCTCCCCC GGTGGCGCGC ACCCGCGCGC GCACACTCGC ACACCGCACC TCAGCTCCTG 360 CCCGGCCTCG CCCCCACCCG CGAGCGCCGA ACCTCCTGGG GCCGGATGCC ATG GGT 416 AAC AAC TTC TCC AGT ATC CCC TCG CTG CCC CGA GGA AAC CCG AGC CGC 464 GCG CCG CGG GGC CAC CCC CAG AAC CTC AAA GAC TCT ATC GGG GGC CCC 512 TTC CCC GTC ACT TCT CAC CGA TGC CAC CAC AAG CAG AAG CAC TGT CCG 560 GCA GTG CTG CCC AGC GGG GGG CTC CCA GCG ACG CCG CTG CTC TTC CAC 608 CCG CAC ACC AAG GGC TCC CAG ATC CTC ATG GAC CTC AGC CAC AAG GCT 656 GTC AAG AGG CAG GCC AGC TTC TGC AAC GCC ATC ACC TTC AGC AAC CGC 704 CCG GTC CTC ATC TAC GAG CAA GTC AGG CTG AAG ATC ACC AAG AAG CAG 752 TGC TGC TGG AGC GGG GCC CTG CTG TTC ACC AGC AAG GAC CCG 800 TCC CGC ATC CAC CCT GAC TCG CTG CCC AAG TAC GCC TGC CCC GAC CTG 848 GTG TCC CAG AGT GGC TTC TGG GCC AAG GCG CTG CCT GAG GAG TTT GCC 896 AAT GAG GGC AAC ATC ATC GTCTC GTG GAC AAG AAG GGC CGT GTC 944 TTC CAC CGC ATC AAC GAC TCG GCT GTT ATG CTG TTC TTC AGC GGG GTC 992 CGC ACG GCC GAC CCG CTC TGG GCC CTG GTG GAC GTC TAC GGC CTC ACG 1040 CGG GGC GTC CAG CTG CTT GAT GAG CTG GTG CTC CCG GAC TGT CTG 1088 CGG CCG CGC TCC TTC ACC GCC CTG CGG CGG CCG TCG CTG CGG CGC GAG 1136 GCG GAC GAC GCG CGC CTC TCG GTG AGC CTA TGC GAC CTC AAC GTG CCG 1184 GGC GCG GAC GGC GAG GCG CCG GCC GCC GGC TGC CCC ATC CCG 1232 CAG AAC TCA CTC AAC TCG CAG CAC AGC CGC GCG CTG CCG GCG CAG CTC 1280 GAC GGC GAC CTG CGT TTC CAC GCC CTG CGC GCC GGC GCG CAC GTC CGC 1328 ATC CTC GAC GAG AG ACG GTG GCG CGC GTG GAG CAC GGG CGC GAC GAG 1376 CGC GCG CTC GTC TTC ACC AGC CGG CCC GTG CGC GTG GCC GAG ACC ATC 1424 TTC GTC AAG GTC ACG CGC TCG GGT GGC GCG CGG CCC GGC GCG CTG TCG 1472 TTC GGC ACC ACG TGC GAC CCC GGC ACG CTG CGG CCG GCC GAC CTG 1520 CCT TTC AGC CCT GAG GCC CTG GTG GAC CGC AAG GAA TTC TGG GCC GTG 1568 TGC CGC GTG CCC GGG CCC CTG CAC AGC GGC GAC ATC CTG GGC CTG GTG 16TC GCC GAC GGC GAG CTG CAC CTC AGC CAC AAT GGC GCG GCC GCC 1664 GGC ATG CAG CTG TGC GTG GAC GCC TCG CAG CCG CTT TGG ATG CTC TTC 1712 GGC CTG CAC GGG ACC ATC ACG CAG ATC CGC ATC CTC GGC TCC ACT ATC GCC GAG CGG GGT ATC CCG TCA CTC CCC TGC TCC CCT GCC TCC ACG 1808 CCA ACC TCG CCC AGT GCC CTG GGC AGC CGC CTG TCT GAC CCC TTG CTC 1856 AGC ACG TGC AGC TCT GGC CCT CTG GGT AGC TCT GCT GGT GGG ACA GCC CCC AAT TCG CCA GTG AGC CTG CCC GAG TCG CCA GTG ACC CCA GGT CTG 1952 GGC CAG TGG AGC GAT GAG TGC ACC ATT TGC TAT GAA CAC GCG GTG GAC 2000 ACG GTC ATC TAC ACA TGT GGC CAC ATG TGC CTC TGC TAC GCC TGT GGC 2048 CTG CGC CTC AAG AAG GCT CTG CAC GCC TGC TGC CCC ATC TGC CGC CGC 2096 CCC ATC AAG GAC ATC ATC AAG ACC TAC CGC AGC TCC TAG CCCGTTGCGG 2145 TGGCCCATCC CGCATACCCA TCTTCTCGGG CTTCAGCCCAACCACC

SEQ ID NO: 3 Sequence length: 546 Sequence type: amino acid Topology: linear Sequence type: protein sequence Asp Ser Ile Gly Gly Ser Phe Pro Val Pro Ser His Arg Cys His His 1 5 10 15 Lys Gln Lys His Cys Pro Pro Thr Leu Ser Gly Gly Gly Leu Pro Ala 20 25 30 Thr Pro Leu Leu Phe His Pro His Thr Lys Gly Ser Gln Ile Leu Met 35 40 45 Asp Leu Ser His Lys Ala Val Lys Arg Gln Ala Ser Phe Cys Asn Ala 50 55 60 Ile Thr Phe Ser Asn Arg Pro Val Leu Ile Tyr Glu Gln Val Arg Leu 65 70 75 80 Lys Xaa Thr Lys Lys Gln Cys Cys Trp Ser Gly Ala Leu Arg Leu Gly 85 90 95 Phe Thr Ser Lys Asp Pro Ser Arg Ile His Pro Asp Ser Leu Pro Lys 100 105 110 Tyr Ala Cys Pro Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala 115 120 125 Leu Pro Glu Glu Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val 130 135 140 Asp Lys Lys Gly Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met 145 150 155 160 Leu Phe Phe Ser Gly Val Arg Thr Val Asp Pro Leu Trp Ala Leu Val 165 170 175 Asp Val Tyr Gly Le u Thr Arg Gly Val Gln Leu Leu Asp Ser Glu Leu 180 185 190 Val Leu Pro Asp Cys Leu Arg Pro Arg Ser Phe Thr Ala Leu Arg Arg 195 200 205 Pro Ser Leu Arg Cys Glu Ala Asp Glu Ala Arg Leu Ser Val Ser Leu 210 215 220 Cys Asp Leu Asn Val Pro Gly Ala Asp Gly Asp Asp Gly Ala Pro Pro 225 230 235 240 Ala Gly Cys Pro Ile Pro Gln Asn Ser Leu Asn Ser Gln His Ser Arg 245 250 255 Ala Leu Pro Ala Gln Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg 260 265 270 Ala Gly Ala His Val Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Leu 275 280 285 Glu His Gly Arg Asp Glu Arg Ala Leu Val Phe Thr Ser Arg Pro Val 290 295 300 Ser Val Ala Glu Thr Ile Phe Ile Lys Val Thr Arg Ser Gly Gly Gly 305 310 315 320 Arg Glu Gly Ala Leu Ser Phe Gly Val Thr Thr Cys Asp Pro Gly Thr 325 330 335 Leu Arg Pro Ala Asp Leu Pro Phe Ser Pro Glu Ala Leu Val Asp Arg 340 345 350 Lys Glu Phe Trp Ala Val Cys Arg Val Pro Gly Pro Leu His Ser Gly 355 360 365 Asp Ile Leu Gly Leu Val Val Asn Ala Asp Gly Glu Leu His Leu Ser 370 375 380 His Asn Gly Ala Ala Al a Gly Met Gln Leu Cys Val Asp Ala Ser Gln 385 390 395 400 400 Pro Leu Trp Met Leu Phe Ser Leu His Gly Ala Ile Thr Gln Val Arg 405 410 415 Ile Leu Gly Ser Thr Ile Met Thr Glu Arg Gly Gly Pro Ser Leu Pro 420 425 430 Cys Ser Pro Ala Ser Thr Pro Thr Ser Pro Ser Ala Leu Gly Ile Arg 435 440 445 Leu Ser Asp Pro Leu Leu Ser Thr Cys Gly Ser Gly Pro Leu Gly Gly 450 455 460 Ser Ala Gly Gly Thr Ala Pro Asn Ser Pro Val Ser Leu Pro Glu Pro 465 470 475 480 480 Pro Val Thr Pro Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys 485 490 495 Tyr Glu His Ala Val Asp Thr Val Val Ile Tyr Thr Cys Gly His Met Cys 500 505 510 Leu Cys Tyr Ser Cys Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys 515 520 525 Cys Pro Ile Cys Arg Arg Pro Ile Lys Asp Ile Ile Lys Thr Tyr Arg 530 535 540 Ser Ser 545

SEQ ID NO: 4 Sequence length: 1641 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence GAC TCC ATC GGG GGC TCC TTC CCG GTG CCC TCT CAC CGA TGC CAT CAC 48 AAG CAG AAG CAT TGC CCG CCT ACG CTG TCA GGT GGG GGG CTC CCG GCC 96 ACG CCG CTG CTC TTC CAC CCC CAC ACT AAG GGC TCC CAG ATC CTC ATG 144 GAC CTC AGC CAC AAG GCC GTC AAG AGG CAGCC AGC TTC TGC AAT GCC 192 ATC ACC TTC AGT AAC CGC CCG GTG CTC ATC TAC GAG CAA GTC AGG CTG 240 AAG NTC ACC AAG AAG CAA TGC TGC TGC TGG AGC GGG GCC CTG CGA CTT GGC 288 TTC ACC AGC AAG AAG GAC CCT TCC CTC ATC CCC GAC TCG CTG CCC AAG 336 TAC GCC TGC CCT GAC CTG GTG TCT CAG AGT GGC TTC TGG GCC AAA GCA 384 TTG CCT GAG GAG TTT GCC AAC GAG GGC AAC ATC ATT GCC TTC TGG GTG 432 GAC AAG AAG GGC CGC GTC TTCTAC ATC AAT GAG TCA GCT GCT ATG 480 CTT TTC TTC AGT GGG GTC CGG ACG GTG GAC CCG CTC TGG GCC CTG GTG 528 GAC GTC TAC GGC CTC ACG CGG GGT GTC CAG CTG CTA GAC AGC GAG CTG 576 GTG CTG CCC GAC TGC CTG CGG CCG CGC TCC TTC ACC GCG CTG CGG CGG 624 CCG TCG CTG CGG TGC GAG GCG GAT GAA GCG CGC CTG TCG GTG AGC CTG 672 TGC GAC CTC AAC GTG CCG GGA GCC GAC GGC GAC GAC GCA CCG CCT 720 GCC GGC TGC CCG ATC CCG CAG AAC TCG CTC AAT TCT CAG CAC AGC CGC 768 GCG CTG CCG GCG CAG CTC GAC GGC GAC CTG CGC TTC CAC GCG CTT CGC 816 GCC GGC GCG CAC GTC CGC ATC CTG GAC GAG AGA GTG GCG CGC CTG 864 GAG CAC GGG CGC GAC GAG CGC GCG CTC GTC TTC ACC AGC CGG CCT GTG 912 AGC GTG GCC GAG ACC ATC TTC ATC AAG GTC ACG CGC TCG GGC GGG GGG 960 CGA GCG GGC GCG CTG TCC TTC GGG GTC ACC TGT GAC CCT GGC ACG 1008 CTG CGG CCC GCG GAC CTG CCC TTC AGC CCC GAG GCC CTG GTG GAC CGC 1056 AAG GAG TTC TGG GCG GTG TGT CGC GTG CCC GGG CCT CTG CAC AGC GGC 1104 GAC ATC CTG GGC CTG GTG GTC ACG GGA GAG CTG CAC CTG AGT 1152 CAC AAC GGC GCG GCG GCC GGC ATG CAG CTG TGC GTG GAT GCC TCG CAG 1200 CCC CTC TGG ATG CTC TTC AGC CTG CAT GGC GCC ATC ACG CAG GTC CGC 1248 ATC CTC GGC TCC ACC ATC ATG AC T GAA CGG GGT GGC CCA TCT CTC CCC 1296 TGC TCA CCT GCC TCC ACT CCA ACC TCA CCC AGT GCC CTG GGC ATC CGC 1344 CTC TCT GAC CCC CTG CTC AGC ACC TGC GGT TCT GGG CCC CTA GGT GGC 1392 TCT GCT GGA GGA GCC CCC AAC TCA CCT GTG AGC CTG CCC GAG CCA 1440 CCG GTG ACC CCA GGT CTG GGC CAG TGG AGT GAT GAA TGC ACC ATT TGC 1488 TAT GAA CAC GCA GTG GAT ACA GTC ATC TAC ACG TGT GGC CAC ATG TGC 1536 CTG TGC TAC TCC GGC CTG CGC CTC AAG AAG GCC CTG CAC GCC TGC 1584 TGC CCC ATC TGC CGT CGC CCC ATC AAG GAC ATC ATC AAG ACC TAC CGC 1632 AGC TCC TAG 1641

SEQ ID NO: 5 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence TAGACGTCCA CCAGGGCCCA GAC 23

SEQ ID NO: 6 Sequence length: 23 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid sequence CGGAGCACTC TCCACGACTC TAT 23

SEQ ID NO: 7 Sequence length: 23 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid sequence AGCACTGCCG GACAGTGCTT CTG 23

SEQ ID NO: 8 Sequence length: 23 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid sequence GTGGTGGCAT CGGTGAGAAG TGA 23

SEQ ID NO: 9 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence ATTAACCCTC ACTAAAG 17

SEQ ID NO: 10 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence GGCCGTGTCT TCCACCGCAT CAA 23

SEQ ID NO: 11 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence CTCGGCTGTT ATGCTGTTCT TCA 23

SEQ ID NO: 12 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Sequence AATACGACTC ACTATAG 17

SEQ ID NO: 13 Sequence length: 23 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid sequence GTGGACGCCT CGCAGCCGCT TTG 23

SEQ ID NO: 14 Sequence length: 23 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid sequence CGATGAGTGC ACCATTTGCT ATG 23

SEQ ID NO: 15 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence AGAGCAGCAG AGGTGGCTGC ACT 23

SEQ ID NO: 16 Sequence length: 23 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid sequence GGCTTGTTCC TCAGCTGGGA CTG 23

SEQ ID NO: 17 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence GACTCCATCG GGGGCTCCTT CCC 23

SEQ ID NO: 18 Sequence length: 23 Sequence type: Number of nucleic acid chains: Single stranded Topology: Linear Sequence type: Other nucleic acid Sequence CTAGGAGCTGCGGGTAGGTCT TGA
23

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI G01N 33/53 G01N 33/53 D // (C12N 15/09 ZNA C12R 1:91) (C12P 21/02 C12R 1:19)

Claims (12)

[Claims] 1. A protein comprising an amino acid sequence having at least the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. A protein comprising an amino acid sequence having at least the amino acid sequence of SEQ ID NO: 3 in the sequence listing. 3. A protein comprising an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. 4. A protein comprising an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of SEQ ID NO: 3 in the sequence listing. A polynucleotide encoding the protein according to any one of claims 1 to 4. 6. A polynucleotide which is a part of the polynucleotide according to claim 5, wherein the polynucleotide comprises 12 or more consecutive bases. 7. An antisense polynucleotide comprising the nucleotide sequence of the antisense strand of the polynucleotide according to claim 5, or a part of a derivative of the antisense polynucleotide, comprising at least 12 consecutive bases. Polynucleotide. 8. The polynucleotide according to claim 5, which is chemically modified. 9. A method for obtaining a cDNA which is a homolog of a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing or SEQ ID NO: 4 in the sequence listing, wherein the method comprises the steps of:
The polynucleotide comprising the coding region of the polynucleotide according to any one of the above as a probe, cD
A method for obtaining a cDNA that hybridizes with the polynucleotide used as the probe from an NA library. 10. A homologue of a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing or SEQ ID NO: 4 in the sequence listing, obtained by the method according to claim 9.
NA. 11. A homolog of the protein according to claim 1 or 2, wherein the protein comprises the amino acid sequence encoded by the cDNA according to claim 10. 12. An antibody that recognizes the protein according to claim 1, 2, 3, 4, or 11.