CA2309336A1 - Neuralized protein, polynucleotide encoding the same, and antibody recognizing the same - Google Patents

Abstract

A gene causative of nerve mutation located in a site in the human tenth chromosome frequently suffering from deletion and having been identified; a protein (a nerve mutation factor) encoded by this gene; and an antibody against this protein. Because of showing a high homology with a Drosophila neuralized gene, this human gene causative of nerve mutation is a human homolog of the neuralized gene. Also, provision is made of a mouse neuralized gene; a protein encoded by this gene; and an antibody against the same.

Description

DESCR=QTION
NEURALIZED PROTEINS, POLYNUCLEOTIDES CODING THE
PROTEINS, AND ANTIBODIES RECOGNIZING THE PROTEINS
TECHNICAL FIELD
This invention relates to homologous proteins of the neuralized proteins of Drosophila as well as to polynucleotides encoding the homologous proteins. It also relates to antisense polynucleotides of said polynucleotides. Additionally, it relates to antibodies recognizing the homologous proteins.
BACKGROUND ART
Among vertebrate animals, human beings are living things that have the most highly differentiated nervous system. Its analysis, however, lags behind due to the complicated mechanism. Especially, concerning tumors that develop in the cerebral nervous system, their analysis has been behind those of other tumors. In the cerebral nervous system, even benign tumors, not to mention malignant ones, may sometimes be life threatening to patients depending on the locations at which the tumors develop. Therefore, progress in elucidation of the development mechanism of cerebral nerve tumor has been awaited.
Up to the present time, it has been known that tumors in the cerebral nervous system, especially, the abnormality in malignant glioma arise from aberration of Chromosome 10. However, the relationship between the actual aberration such as the deficiency of Chromosome 10 and clinical symptoms has not been clarified.
On the other hand, in Drosophila, many mutants have been obtained during the analysis of their neural development, and the genetic analysis thereof has advanced. As a result, the following have, thus far, been suspected to be causative genes for the mutations:
"Notch," "Delta," "Big Brain," "Neuralized," "Master Mind," "Almondex," and "Enhancer of Split," etc.
Among the aforementioned genes, human homologues have been acquired with respect to "Notch," "Delta,"
and "Enhancer of Split."
DISCLOSURE OF INVENTION
With a view to elucidating tumors in the cerebral nervous system and the aberration of Chromosome 10, an object of this invention is to identify sites in Chromosome 10 at which the frequency of deficiency is high, to identify causative genes for nerve mutation, and to provide said genes. It is also an object of the invention to provide proteins (nerve-mutating factor) that the genes encode and further to provide antibodies against the proteins.
Specifically, this invention provides a human nerve mutating causative gene which is present at the sites which are missing with high frequency in human Chromosome 10. It also provides a murine nerve mutating causative gene having homology with the human nerve mutating causative gene. These genes are highly homologues to the neuralized gene of Drosophila and are, respectively, human and murine homologues of the neuralized gene. This invention has revealed that the neuralized gene previously known in Drosophila also exists in vertebrate animals, more specifically mammals such as human and mouse.
The invention also provides antisense polynucleotides comprising base sequences that are complementary to the aforementioned base sequences.
In addition, this application discloses a method for producing a neuralized protein. Specifically, a method is disclosed whereby a transformant to which neuralized cDNA has been introduced is allowed to produce a neuralized protein. The neuralized proteins produced by the method are also disclosed.
Additionally, there are disclosed a method for producing a variant of the neuralized protein where one or more amino acids within the amino acid sequence of the neuralized protein have been substituted, deleted or subject to addition, as well as the variant of the neuralized protein produced by the production method.
Further, this invention discloses antisense polynucleotides comprising base sequences that are complementary to polynucleotides encoding the neuralized proteins or to polynucleotides encoding variants of the neuralized proteins. Although antisense polynucleotides are embraced by polynucleotides, they are referred to as "antisense polynucleotide(s)" in this specification particularly where the polynucleotides comprising base sequences for an antisense strand are clearly meant. Representatives of the antisense polynucleotide are antisense DNAs and antisense RNAs.
Still further, the invention discloses a polynucleotide that is a part, which comprises not less than 12 bases, or the whole of a polynucleotide encoding the neuralized protein or a variant thereof.
This polynucleotide, if it is a part of the coding region, can be used in producing a partial length protein of the neuralized protein or of a variant of the neuralized protein. It can also be used as a probe.
Further, the invention discloses an antisense polynucleotide that is a part, which comprises not less than 12 bases, or the whole of an antisense polynucleotide,of the neuralized protein or a variant thereof.
This antisense polynucleotides is able to respectively inhibit biosynthesis of the neuralized protein or that of the variant of the neuralized protein. It can also be used as a probe.
Furthermore, the invention discloses polynucleotides that are obtained by chemically modifying the aforementioned polynucleotides, including antisense polynucleotides.
Moreover, the invention discloses that through an analysis by the northern blot hybridization using DNA
probes, mRNA of the neuralized can be detected in various tissues or cell strains of human or mouse and that mRNA of the neuralized is naturally expressed in these animals.
In addition, the invention discloses a human homologous protein of the neuralized protein encoded by a human homologous gene of the neuralized gene: it is also a homologous protein of the Drosophila neuralized protein. Here, in this specification the neuralized proteins in various vertebrate animals are indicated collectively where no names of the animals, from which the proteins are derived, are mentioned. The aforementioned neuralized proteins can be produced by transformants to which the neuralized genes have been introduced.

The invention encompasses a method whereby a polynucleotide encoding the human neuralized, a polynucleotide encoding the murine neuralized, or a part of the foregoing comprising not less than 12 bases is used as a probe to obtain a neuralized cDNA from a cDNA library of any other vertebrate animal, preferably a mammal, as well as encompasses the mammalian neuralized cDNA obtained by the method.
Here, the probes that can be used are a cDNA
fragment comprising a base sequence of high homology portion between the base sequence of the human neuralized cDNA and the base sequence of the murine neuralized cDNA, a polynucleotide obtainable from the base sequence of said cDNA fragment, including single-stranded DNA (which contains antisense strands) and RNA
against the cDNA or chemically modified ones thereof.
If the probes are allowed to hybridize with the cDNA
libraries of other animals, neuralized cDNAs of said animals could be acquired.
Further, the invention provides neuralized proteins that are homologous proteins of the human neuralized protein and the murine neuralized protein which the aforementioned cDNAs (the human neuralized cDNA and the murine neuralized cDNA) encode.
Still further, the invention provides antibodies that recognize the aforementioned neuralized proteins and variants thereof. In this application, there is disclosed that the antigenicity of the neuralized proteins, namely antibodies, can be prepared from the neuralized proteins.
Additionally, the invention discloses the locations of the human neuralized gene on its chromosomes.
BRIEF DESCRPTION OF DRAWINGS
Fig. 1 is a photograph of electrophoresis showing the results of analysis of expression of the neuralized gene with respect to individual human tissues according to northern blot hybridization.
Fig. 2 is a photograph of electrophoresis showing the results of analysis of expression of the neuralized gene with respect to a normal human cerebral tissue (three samples), an astrocytoma tissue (anaplastic astrocytoma: one sample, and glioblastoma: three samples), and glioma cell strains according to northern blot hybridization.
Fig. 3 is a photograph of electrophoresis showing the results of analysis of expression of the neuralized gene with respect to a normal human cerebral tissue, medulloblastoma, and glioma cell strains according to RT-PCR .

BEST MODE FOR CARRYING OUT THE INVENTION
The homologous protein of the Drosophila neuralized protein according to this invention is not limited to a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing or in SEQ ID NO: 3 in the Sequence Listing. The invention encompasses proteins encoded by cDNAs obtainable from the cDNA libraries of various animals: the probes used to obtain the cDNAs are polynucleotides encoding those proteins, polynucleotides comprising parts of the coding regions of the foregoing, especially the base sequences set forth in SEQ ID NO: 2 or in SQ ID NO: 4 in the Sequence Listing or partial base sequences of the coding regions of the base sequences.
A highly conserved base sequence between the human neuralized gene and the murine neuralized gene is, for example, a sequence from G at position 576 to C at position 938 of the base sequence of the human neuralized cDNA set forth in SEQ ID N0: 2 in the Sequence Listing: it corresponds to from the 82nd base to the 446th base of the base sequence of the murine neuralized cDNA set forth in SEQ ID NO: 4 in the Sequence Listing.
DNA comprising this base sequence can be used as a probe to obtain the full-length neuralized gene from the cDNA library of an objective animal, following the manipulations described below.
(1) DNA which serves as the probe is labeled with a Takara MEGA LABEL kit (Registered Trademark:
available from Takara Shuzo Co. Ltd.) as exactly S described in its manual.
(2) Next, a DNA reaction solution having the composition described below is prepared; and after incubating this solution at 37 ° C for 30 min, it was subjected to heat treatment at 70 ° C for 10 min, causing the enzyme to be deactivated.
DNA probe ( 2pmo1/,(.cl ) 2 ,ul 10-Fold phosphorylation buffer 2 ,u 1 ~ ~,-32P~ATP (370 MBq/ml) 5 ,ul (available from Amersham Pharmacia Biotech Inc.) T4 polynucleotidekinase 1 ,u l Total 10 ,u 1 (3) A SEPHADEX G-25 (Registered Trademark:
available from Amersham Pharmacia Biotech Inc.) equilibrated with TE50 (50 mM Tris-HC1, pH 8.0, 1 mM, EDTA) is packed into a 1.5-ml Polyprep column (available from Bio-Rad Laboratories Inc.) to give a bed volume of 1 ml. The DNA reaction solution that has been heat-treated in step (2) is loaded onto the column.
(4) Subsequently, the column is eluted with 200 ,c.c 1 of T50E four times, and a labeled DNA probe is obtained from the fraction eluted by the second 200 ,u 1.
(5) The above-obtained labeled DNA probe fraction of 150,u 1 is hybridized to the cDNA probe of a plaque fixed on a nitrocellulose membrane under the conditions described below: Fixation of a cDNA library onto a nitrocellulose membrane can be carried out according to the method as described in Molecular Cloning, 2nd ed.;
Cold Spring Harbor Laboratory Press: 1989; 9.38-9.40.
(i) Prehybridization 6xSSC
5xDenhaldt~s 0.05$ sodium pyrophosphate 100 ,u g/ml denatured herring sperm DNA
0.5% SDS
Total amount of solution: 50 ml Reaction temperature: 37 ° C
Reaction time: 1 hr (ii) Hybridization 6xSSC
lxDenhaldt~s 0.05$ sodium pyrophosphate 100 ,u g/ml denatured herring sperm DNA
1x106 cpm/ml cDNA probe Total amount of solution: 50 ml Reaction temperature: 42 ° C
Reaction time: 18 hr (6) The nitrocellulose membrane having undergone hybridization is once washed under each of the following conditions:
(i) 6xSSC, 0.1% SDS 500 ml Temperature: 40 ° C
Time: 20 min (ii) 3xSSC, 0.1% SDS 500 ml Temperature: 42 ° C
Time: 20 min (7) The washed nitrocellulose membrane is exposed to an X-ray film such as Kodak XR-5 film at -80 ° C
overnight and an autoradiograph is taken.
(8) The positions of positive plaques are determined from the resultant autoradiograph and the corresponding plaques on agar are recovered into SM
solution.
(9) The recovered plaques are allowed to undergo plaque formation again on a NZY agar medium according to a standard method and they are fixed on a nitrocellulose membrane.

(10) Steps (5)-(9) are repeated three times to make the positive plaques into a single one. The plaque is then recovered and suspended in 100 ,ul of SM
solution to stabilize the phage. cDNA that will be isolated from the plaque is the neuralized cDNA.
3. Large Scale Preparation of the Neuralized cDNA
(1) The phage of plaque suspended in SM solution, 50 ,u l, is mixed with 20 ,u l of Y1090r E. coli and allowed to stand at 37 ° C for 15 min.
(2) Subsequently, the solution mixed in step (1) is transferred to a 10-ml NZY medium containing 100 ,cc g/ml ampicillin and culturing is carried out at 37 ° C
for 6 h.
(3) Centrifugation is carried out at 8,000 rpm for 5 min, and the supernatant is recovered.
(4) To the supernatant is added 1 ml of 5 M NaCl and 1.1 g of polyethylene glycol 6000, achieving their dissolution.
(5) The solution is placed on ice for 1 h, and then, centrifugation is carried out at 10,000 rpm at 4 ° C for 20 min.
(6) The precipitate is recovered and is suspended in 700 ,ul of SM solution.
(7) To this is added 500 ,u 1 of chloroform and stirring is done to dissolve the remaining E. coli.
(8) Centrifugation is carried out at 5,000 rpm for 10 min and the aqueous layer is recovered.
(9) To this is added each 1 ,(.C1 of RNaseA (1 mg/ml) and Dnasel (5 mg/ml) (both available from Sigma).
After allowing to stand at 37 ° C for 1 h, 600 ,(.cl of 20% polyethylene glycol 6000 (0.8 M NaCl) is added and it is allowed to stand on ice for 30 min.
(10) Centrifugation is carried out at 15,000 rpm at 4 ° C for 20 min and the precipitate is recovered.
(11) To this precipitate are added 500 ,ccl of SM
solution, 50 ,c.~l of 5M NaCl, and 50 ,ul of 0.5 M EDTA.
Further, 400 ,u 1 of phenol is added and stirring is done to dissolve the phage and to liberate the cDNA.
(12) The solution is centrifuged at 15,000 rpm for 5 min, and then, the aqueous layer is recovered. To this is added 1 ml of ethanol, centrifugation is carried out at 15,000 rpm for 20 min, and the liquid layer is discarded.

(13) The precipitate is washed with 1 ml of 70~
ethanol and is dissolved in 100 ,ul of TE solution (10 mM Tris-HC1, pH 8.0, 1 mM EDTA) to obtain a DNA
solution.
4. Determination of the Base Sequence of Neuralized CDNA
The full base sequence of the neuralized cDNA is determined using an autosequencer, according to the dyeterminator method.
When the result of sequencing reveals that a DNA
containing the full length of the coding region (which hereinafter may be referred to as "the full-length DNA") has not been obtained, a combination of DNAs is selected so as to cover the coding region, and the DNAs are cut at the suitable restriction enzyme sites, which are contained in their overlapping parts, and are linked together, whereby the full-length DNA can be obtained. Alternatively, primers are designed in such a manner as to contain the full length of the coding region. The primers are then used in performing PCR
with the template of a cDNA library from an objective vertebrate animal, preferably a mammal: this can result in obtaining the full-length DNA.
5. Determination of the Amino Acid Sequence From the base sequence determined in step 4, the amino acid sequence of the neuralized cDNA is to be determined.
6. Preparation of Transformants Neuralized cDNAs obtained above in a similar manner to step 9 in Example 1 are inserted into suitable vectors such as a TA cloning vector, and then, the vector can be introduced into a host such as E.
coli to prepare a transformant.
Natural or artificial mutations make it possible to alter a polynucleotide without changing the principal function of a polypeptide that the polynucleotide encodes. (For example, the method as described in Molecular Cloning, 2nd ed.: 15.1-15.113.) Concerning the neuralized of this invention, this method also allows the preparation of a protein comprising an amino acid sequence obtained by making substitution, deletion or addition of one or more amino acids in the amino acid sequence set forth in SEQ ID
NO: 1 or in SEQ ID NO: 3 in the Sequence Listing:
namely, a variant of the neuralized protein.
When the variants are prepared by the site-directed mutagenesis technique, a single operation can normally substitute, delete, or add several amino acids.
Repetition of this operation plural times can further substitute, delete, or add more amino acids, and thus, desirable variants can be prepared. It is preferred that homology between the neuralized of this invention and its variant be not less than 75% at an amino acid level; more preferably not less than 90% and most preferably not less than 95%.
The amino acid sequences for variants of the neuralized proteins according to this invention can be determined by the base sequences of genes encoding said variants. For example, it is feasible through commercially available programs such as GENETIX-MAC
(Registered Trademark: available from Software Development Inc.).
Because of degeneracy in genetic codes, it is possible to substitute at least a part of bases in the base sequence of a polynucleotide with other kinds of bases without altering the amino acid sequence of a polypeptide produced by the polynucleotide. Therefore, by a polynucleotide of this invention encoding the neuralized protein is meant that which contains all patterns of degeneracy.
DNA comprising the base sequence set forth in SEQ
ID NO: 2 in the Sequence Listing is the human neuralized cDNA existing in nature; and DNA comprising the base sequence set forth in SEQ ID NO: 4 in the Sequence Listing is the murine neuralized gene existing in nature.
This invention encompasses polynucleotides and derivatives thereof comprising the base sequences of antisense strands for the coding regions of polynucleotides that encode the neuralized proteins.
The antisense polynucleotide is able to hybridize to a polynucleotide encoding the neuralized, and if the polynucleotide to which it is hybridized is one of the coding region, it is able to inhibit the biosynthesis of a polypeptide that said polynucleotide encodes.
It is preferred that the antisense polynucleotide for the inhibition of biosynthesis of the polypeptide comprise not less than 15 bases. On the other hand, when a full-length polynucleotide is to be incorporated into cells, it is unsuitable if its length is too long.
In cases where an antisense polynucleotide is to be incorporated into cells and is allowed to inhibit biosynthesis of the neuralized protein, it is advisable that an antisense polynucleotide be used which comprises not less than 12 bases but not more than 30 S bases, preferably not less than 15 bases but not more than 25 bases, and more preferably not less than 18 bases but not more than 22 bases.
The antisense polynucleotides of this invention or parts thereof encompass all of those in which a plurality of nucleotides comprising bases, phosphoric acid, and sugars are bonded, including those not present in nature; their representatives are antisense DNAs and antisense mRNAs.
With respect to the antisense polynucleotides of this invention, a variety of derivatives can be obtained by using the antisense technology known in the art, which have enhancement in binding strength with objective DNAs or mRNAs, tissue selectivity, cell permeability, nuclease resistance, and intracellular stability.
In view of the ease of hybridization, it is thought to be advantageous that antisense polynucleotides or derivatives thereof having the base sequences complementary to those of the regions which form stem loops be designed. The antisense polynucleotides of this invention or derivatives thereof are capable of forming stem loops if necessary.
It is also expected that such polynucleotides as having sequences complementary to those in the vicinity of the translation start codon or at a ribosomal binding site, the capping site or a splicing site are generally provided with a great expression inhibitory effect. Thus, the great expression inhibitory effect is expected for the antisense polynucleotides or derivatives thereof according to this invention that contain the genes encoding the neuralized or that contain sequences complementary to those of mRNAs in the vicinity of the translation start codon, or at a ribosomal binding site, the capping site or a splicing site.
At present, generally known derivatives are preferably those which have enhancement in at least one of nuclease resistance, tissue selectivity, cell permeability, and binding strength. Most preferably, the polynucleotide derivatives are those which have phosphorothioate bonds as their skeleton structures.
The polynucleotides or derivatives thereof according to this invention also encompass derivatives having these functions and structures.
If the antisense polynucleotides of this invention are of the natural type, they can be prepared either by synthesis using a chemical synthesizer or by the PCR

method which employs the genes encoding the neuralized as templates. Also, among the derivatives, there are ones that can be chemically synthesized, such as those of the methylphosphonate type and the phosphorothioate type. In these cases, operations are conducted following the manual attached to the chemical synthesizer and the resultant synthetic products are purified by a HPLC method using reverse phase chromatography or the like; thus, the objective antisense polynucleotides or derivatives thereof can be obtained.
The polynucleotides encoding the neuralized, antisense polynucleotides thereof, or polynucleotides being parts of the foregoing (polynucleotides comprising base sequences of not less than 12 consecutive bases) according to this invention can be used as probes for screening neuralized genes from cDNA
libraries and the like. Where the species of an objective animal is the same as the species of an animal from which the polynucleotide that serves as the probe has been derived, portions in the non-coding region can even be used. Here, those having GC
contents of from 30 to 70~ can preferably be used.
Especially preferred are polynucleotides comprising base sequences of not less than 15 consecutive bases.
Said polynucleotides to be used as probes may be derivatives thereof. It is normally recognized that sequences having base numbers greater than those described above are those having specificities. with respect to cDNA libraries that are used in screening with the aid of said probes, those prepared from mRNAs can preferably be used. A group of cDNAs selected from these cDNA libraries by random sampling may be employed as samples for screening. Commercially available cDNAsv are also usable.
For example, DNA having a base sequence comprising 12 or more consecutive bases within the base sequence set forth in SEQ ID NO: 2 or in SEQ ID NO: 4 in the Sequence Listing, or a polynucleotide (antisense polynucleotide) capable of hybridizing to said DNA can be used as the probe to screen the neuralized gene from a cDNA library or the like.
The polynucleotides encoding the neuralized, antisense polynucleotides thereof, or polynucleotides comprising parts of the foregoing according to this invention are used as the probes to carry out northern blot hybridization with respect to mRNAs derived from various tissues: this enables the detection of any tissue where mRNA derived from the neuralized gene has expressed.
In the chemical synthesis of DNAs or RNAs, chemical modification such as methylation or biotinylation of their side chains and substitution of O of their phosphoric acid group portions with S is well known. For example, when the DNA set forth in SEQ
ID N0: 2 in the Sequence Listing is chemically synthesized, it is possible to carry out the chemical modification and to synthesize a DNA different from the DNA shown in the Sequence Listing per se.
cDNAs can also be labeled with radioisotopes even if they are obtained from cDNA libraries.
Accordingly, DNAs and RNAs of this invention encompass the chemically modified DNAs, RNAs, and antisense polynucleotides within their scope. The chemically modified DNAs or RNAs are able to exert both the function of encoding proteins and the function as probes; the chemically modified antisense polynucleotides are able to exert both the function of inhibiting the biosynthesis of probes or proteins and the function as probes.
According to methods known in the art, it is possible to introduce plasmids into suitable hosts such as E. coli and then to obtain transformants.
Transformants to which the neuralized genes of this invention have been introduced are grown and the genes are amplified, or proteins are allowed to express to prepare the neuralized proteins or variants thereof, which steps are all possible. Subsequently, the prepared products are recovered, and if necessary, manipulations such as concentration; solubilization, dialysis, and various chromatographic techniques are conducted; thus, it is possible to purify the neuralized proteins or variants thereof according to this invention.
A variety of textbooks are available for the cultivation of transformants. According to methods known in the art, it is also possible to prepare the neuralized proteins or variants thereof on the basis of the base sequences as described in this invention. At this juncture, any of bacteria such as E. coli, yeast, and animal cells can be used as a host. Particularly, animal cells are preferable. For the introduction of genes into cells, the liposome method, electroporation, and the like can be used. Particularly, it is preferable to use nuclear microinjection.
The methods for purifying the neuralized proteins or variants thereof from the resultant cultured products include immunoprecipitation, salting out, ultrafiltration, isoelectric precipitation, gel filtration, electrophoresis, a variety of affinity chromatography (e. g., ion-exchange chromatography, hydrophobic chromatography and antibody chromatography), chromatofocusing, adsorption chromatography, and reverse phase chromatography; and purification may be carried out by selecting them as appropriate.
Also, in the production stage the neuralized proteins or variants thereof to be produced may be prepared by transformants as fusion peptides with other polypeptides. In this case, the purification step requires a manipulation that slices out the neuralized proteins or variants thereof through treatment with a chemical substance such as bromocyanogen or with an enzyme such as protease.
As Example 3 illustrates the antigenicity of the neuralized protein, this invention reveals the following: an antibody can be readily obtained by immunizing an animal, other than human and the animal from which the neuralized protein is derived, with the neuralized protein produced by the aforementioned method or with an oligonucleotide comprising an amino acid sequence that is specific for the neuralized protein. Consequently, the antibodies of the invention that recognize the neuralized proteins (which hereinafter may be referred to as "the neuralized antibodies") encompass, within their scope, those which can be obtained by immunosensitization of animals, other than human and the animals from which the neuralized proteins are derived, with the neuralized proteins and which can be confirmed to recognize the neuralized proteins by western blotting, ELISA, immunostaining (e. g., staining tissues of lyophilized specimens or paraffin specimens), or the like.
For an immunogen, part of a protein is used although it is conjugated to other carrier protein such as bovine serum albumin, which is a well-known method.
The part of a protein may be synthesized using a peptide synthesizer. Here, the part of a protein is preferably eight amino acid residues or more.
It is well known that if polyclonal antibodies against substances which have manifested their antigenicity can be obtained by immunosensitization, monoclonal antibodies are produced by the use of hybridomas employing lymphocytes of the immunized animals. (Antibodies; A Laboratory Manual; Cold Spring Harbor Laboratory Press: 1988; Chapter 6.) Accordingly, the neuralized antibodies of this invention encompass monoclonal antibodies within their scope.
In this invention, the antibodies encompass active fragments thereof. The term "active fragments" means the fragments of antibodies that possess antigen-antibody reactivity; and specifically named are F(ab')2, Fab', Fab, Fv, etc.
For example, F(ab')2 results if an antibody of this invention is digested with pepsin, and Fab results if digested with papain. F(ab')z is reduced with a reagent such as 2-mercaptoethanol and alkylated with monoiodoacetic acid to give Fab'. Fv is an active fragment of monovalent antibody obtained by binding the heavy chain variable region and the light chain variable region through a linker.
Chemeric antibodies are obtained by retaining these active fragments and substituting other parts with fragments from other animals.
The antibodies of this invention can be used in the elucidation of functions such as expression of the neuralized as well as in the elucidation of formation mechanism of malignant tumors.
For the detection of the neuralized, mentioned are methods using antibodies and those utilizing enzymatic reactions.
For the method using antibodies, specifically mentioned are as follows: (1) a technique to detect the neuralized by the use of a labeled neuralized antibody;
and (2) a technique to detect the neuralized by the use of a neuralized antibody or a secondary labeled antibody thereof. As the label, radioisotopes (RI), enzymes, avidin, biotin, and fluorescent substances such as FITC and Rhodamine are to be utilized, for example.
For the method utilizing enzymatic reaction, mentioned are, for example, the identification technique of immune reaction molecules which employs ELISA, immunoagglutination, and western blotting, and those analogous to the foregoing.
EXAMPLES
Hereinbelow, this invention will be illustrated in more detail, but it should not be limited to the following examples.

I. Identification of the Position of Deficiency of Chromosome 10 in Malignant Glioma (1) Preparation of Cells Forty-six cases of human malignant glioma tissue and peripheral blood of malignant glioma patients were collected. The peripheral blood was collected in 5 cc into blood-collecting tubes with the addition of EDTA, and leukocyte nuclear cells were recovered by specific gravity centrifugation using FIRECALL-BAKE (available from Amersham Pharmacia Biotech Inc.). With respect to the tissue, tissue sections (5 mm square) removed in operations or samples from biopsy were treated with collagenase and trypsin to prepare cell suspensions.
(2) Preparation of DNAs DNAs were extracted from the resultant cell suspensions using a Xeagen Blood and Cell Culture DNA

kit (available from Xeagen Inc.) according to its protocol as attached.
(3) Microsatellite Analysis The resultant DNAs were analyzed using primers which were dye-labeled microsatellite markers of 16 sites along the long arm of Chromosome 10.
Consequently, in malignant gliomas of grade III
and grade IV frequently noted was the loss of heterogeneity (LHO) of genes in the regions containing D10S540 and D10S566 among the microsatellite markers along the long arm of Chromosome 10.
(4) STS Mapping Then STS mapping around D10S566 was carried out using the panel of whole genome radiation hybrid and YAC clones. The result revealed the presence of D10S566 in 1Oq25, as well as the positional relationship of STS markers in a vicinity thereof.
II. Identification of Neuralized Gene 1. Searching of EST Data Base From the sequences registered (EST) in EST Data Base on Internet (a date base in which the sequences of mRNA fragments have been registered:
http://www.ncbi.nlm.nih.qov./i html), EST existing in the vicinity of from 1Oq24 to 1Oq25 was searched, and H82634 was hit. When the localization of this EST was determined by PCR with the radiation hybrid panel and YAC clones, it was found out that H82634 was positioned between D10S540 and D10S566 on human Chromosome 10. A
possibility thus arose that a gene containing H82634 is the causative gene for malignant glioma.
S 2. Searching of TIGR Data Base From the sequences registered in TIGR Data Base on Internet (a date base in which the sequence of mRNA
fragments have been registered:
http://www.ncbi.nlm.nih.gov./i html), sequences having homology with H82634 were searched for. The result revealed that H82635 was a sequence complementary to H82634. Based on these two sequences, base sequence A
described below (the base sequence set forth in SEQ ID
NO: 23 in the Sequence Listing) was determined.
1S Base Sequence A:
CAGCTGGACG CCCCGNTGNA GGCCGTAGAC GTCCACCAGG GCCCAGAGCG
GGTCGGCCGT GCGGACCCGC TGAAGAACAG CATAACAGCC GAGTCGTTGA
TGCGGTGGAA GACACGGCCC TTCTTGTCCA CCCAGAATGC GATGATGTTG
CCCTCATTGC CAAACTCCTC AGGCAGCGCT NTGGCCCAGA AGCCACTCTG

CGGGACGGGT CCTTGCTGGT GAAGCCAGCC GCAGGCCCCG CTCCAGCACT
GCTTCTTGGT GATCTTCAGC CTGACTTGCT CGTAGATGAG GACCGGGCGG
TTGCTGAAGG TGATGNCGTT GCAGAAGCTG GCCTGCCTCT TGNACAGCCT
TGTGGCTGAG GTCCATGAGG ATCTGGGAGC CCTTGGTGTG CGGGTGGAAG

TTCTGCTTGT GGTGGCATCG GTGAGAAGTG ACGGGGAAGG GGCCCCCGAT

AGAGTCGTGG AGAGTGCTCC GGGTGAT
3. Synthesis of Primers To obtain a DNA fragment of base sequence A
described above, the following primers were synthesized.
Primer P1, 5'-TAGACGTCCA CCAGGGCCCA GAC-3' (the base sequence set forth in SEQ ID NO: 5 in the Sequence Listing), as a 5'-primer and Primer P2, 5'-CGGAGCACTC
TCCACGACTC TAT-3' (the base sequence set forth in SEQ
ID No: 6 in the Sequence Listing), as a 3'-primer were designed and synthesized with a DNA synthesizer (Model 392 available from ABI). The synthesized primers were adjusted to 20 pmol/,ctl with distilled water. The synthesis of primers as will be described below was carried out in the same manner.
4. PCR
Human fetus brain cDNA library (available from CLONTECH Laboratories Inc.) was used as a cDNA library and PCR was performed under the following conditions:
CDNA 0 . 8 ,CC 1 dNTPmix(each 2.5 mM) 0.6 ,CCl Primer P 1 0 . 6 ,u 1 Primer P2 0 . 6 ,tt 1 3.3xPCR Buffer 4.5 ,u.l 25 mM MgClz ~ 0.72 ,ul Distilled water 6.68 ,u 1 Total 14 . 5 ,u 1 Mineral oil (15 ,u 1) was overlaid on the aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,u 1, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 35 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1 min. Finally, the extension reaction of fragment was carried out at 72 °C
for 10 min, thus completing PCR.
After the reaction, the PCR product was subjected to electrophoresis on a minigel of 1.5~ agarose gel.
An about 0.6 kb band that was believed to be the gene encoding the neuralized protein was sliced off, and the PCR product was recovered. Furthermore, a part of the recovered product was again subjected to the minigel electrophoresis described above, which ascertained that a band of about 0.6 kb appeared. This DNA fragment will be referred to as "Fragment A" hereinafter.
4. Incorporation into a Vector Fragment A was subjected to subcloning under the following conditions, using a pCRIITA cloning vector kit (available from Invitrogen Corp.).

Sterilized distilled water 5 ,u 1 lOx Ligation buffer 1 ,ul pCRI I vector 2 ,u 1 DNA fragment 1 ,u 1 T4DNA ligase 1 ,Cll Total 10 ,u l Reaction was carried out at 14 ° C overnight to yield a ligation mixture.
5. Transformation Transformation was performed using a TA cloning kit.
(i) To E. coli cells (50 ,tcl) on ice were added 2 ,ctl of 0.5 M 2-mercaptoethanol and the ligation mixture prepared in step 4 described above, and it was allowed to stand for 30 min. Subsequently, it was incubated at 42 ° C in a hot bath while being shaken at 225 rpm.
(ii) Next, the E. coli cells were spread over a LB
agar plate to which ampicillin, X-Gal, and IPTG had been added. Incubation was done at 37 ° C for 18 h and colonies in white and in blue appeared.
6. Miniscale Culturing Four white colonies were picked up from the aforementioned plate and each colony was put in a tube in which 3 ml of LB medium (ampicillin added) was present. This was shake-cultured at 37 ° C overnight.

7. Miniscale Preparation A Plasmid was prepared according to the alkali method (for example, a method as described in Molecular Cloning; A Laboratory Manual, 2nd ed; 1.25-1.28, vide supra) 8. DNA Sequencing Plasmid (1 ,ccl) prepared in step 7 described above was taken up and diluted with 99 ,(,tl of TE. Absorbance at 260 nm (A260) was measured and DNA values were computed: the computation was based on that the A260 value being 1.0 was taken as 50 ,ccg/ml. The plasmid was diluted with TE buffer so that DNA reached 1 ,u g/ml based on its A260 values. The base sequence of Fragment A was determined by the dye-terminator method, using an autosequencer (Model 373S available from ABI).
9. 5'-Extension On the basis of the base sequence of Fragment A, Primer P3: 5'-AGCACTGCCG GACAGTGCTT CTG-3' (the base sequence set forth in SEQ ID NO: 7 in the Sequence Listing) was synthesized. Employing this primer and a human fetus brain cDNA library as a template, single PCR was performed under the following conditions:
cDNA 1 ,(.l 1 dNTPmix 0 . 6 ,(.L 1 Primer P3 1 ,ccl 3.3x PCR buffer 4.5 ,(.tl 2 5 mM MgC 12 0 . 7 2 ,cc 1 Distilled water 6.68 ,u.l Total 14 . 5 ,u 1 Mineral oil (15 ,c.cl) was overlaid on the aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,u 1, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 50 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1.5 min.
Subsequently, the extension reaction of fragment was carried out at 55 °C for 2 min and finally at 72 °C for 10 min, thus completing PCR.
Next, this PCR product was used as a template and second PCR was performed. Primer P4, 5'-GTGGTGGCAT
CGGTGAGAAG TGA-3' (the base sequence set forth in SEQ
ID NO: 8 in the Sequence Listing) was synthesized.
Employing this primer and Primer T3, 5'-ATTAACCCTC
ACTAAAG-3' (the base sequence set forth in SEQ ID NO: 9 in the Sequence Listing), as a 5'-primer, PCR was performed under the following conditions:
PCR product 1 ,u 1 dNTPmix 1 ,C.ll Primer P4 1 ,u 1 Primer T3 1 ,ul 3.3x PCR buffer 7.5 ,u 1 25 mM MgCl2 1.2 ,cc1 Distilled water 11.8 ,tcl Total 24.5 ,ul Mineral oil (20 ,u 1) was overlaid on the aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,u 1, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 40 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1 min. Subsequently, the extension reaction of fragment was carried out at 55 °C for 2 min and finally at 72 °C for 10 min, thus completing PCR. This DNA fragment will be referred to as "Fragment B" hereinafter.
10. 3'-Extension On the basis of the base sequence of Fragment A, Primer P5: 5'-GGCCGTGTCT TCCACCGCAT CAA-3' (the base sequence set forth in SEQ ID No: 10 in the Sequence Listing) was synthesized. Employing this primer and human fetus brain cDNA library as a template, single PCR was performed under the following conditions:

CDNA 1 ,CL 1 dNTPmix 0 . 6 ,u 1 Primer P5 1 ,ul 3.3x PCR buffer 4.5 ,CL1 25 mM MgCl2 0.72 ,ul Distilled water 6.68 ,ctl Total 14 . 5 ,u 1 Mineral oil (15 ,u 1) was overlaid on the aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,ccl, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 50 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1.5 min. Finally, the extension reaction of fragment was carried out at 72 °C for 10 min, thus completing PCR.
This PCR product was used as a template and second PCR was performed. Primer P6, 5'-CTCGGCTGTT ATGCTGTTCT
TCA-3' (the base sequence set forth in SEQ ID NO: 11 in the Sequence Listing) was synthesized. Employing this primer and Primer T7, 5'-AATACGACTC ACTATAG-3' (the base sequence set forth in SEQ ID NO: 12 in the Sequence Listing), as a 5'-primer, PCR was performed under the following conditions:

PCR Product 1 ,CC 1 dNTPmix 1 ,Cll Primer P6 1 ,ul Primer T7 1 ,u 1 3.3x PCR buffer 7.5 ,u 1 25 mM MgCl2 1.2 ,ul Distilled water 11.8 ,(.tl Total 24.5 ,ul Mineral oil (20 ,u 1) was overlaid on the aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,u 1, (Registered Trademark, available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 40 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1 min. Finally, the extension reaction of fragment was carried out at 72 °C
for 10 min, thus completing PCR. This DNA fragment will be referred to as "Fragement C" hereinafter.
11. DNA Sequencing Direct sequencing was performed on Fragments B and C by the dye-terminator method, relaying on Primer P4 and Primer P6.
12. 3'-Extension On the basis of the base sequence of Fragment C, Primer P7: 5'-GTGGACGCCT CGCAGCCGCT TTG-3' (the base sequence set forth in SEQ ID NO: 13 in the Sequence Listing) and Primer P8: 5'-CGATGAGTGC ACCATTTGCT ATG-3' (the base sequence set forth in SEQ ID NO: 14 in the Sequence Listing) were synthesized. Employing first S Primer P7 and then a human fetus brain cDNA library as a template, single PCR was performed under the following conditions:
CDNA 1 ,Lll dNTPmix 0 . 6 ,(.t l Primer P7 1 ,t,t1 3.3x PCR buffer 4.5 ,ul 2 5 mM MgC 12 0 . 7 2 ,CC 1 Distilled water 6.68 ,(,cl Total 14 . 5 ,u 1 Mineral oil (15 ,(,tl) was overlaid on the aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,u 1, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 50 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1.5 min. Finally, the extension reaction of fragment was carried out at 72 °C for 10 min, thus completing PCR.
This PCR product was used as a template and second PCR was performed. With the use of Primers P8 and T3 PCR was performed under the following conditions:
PCR product 1 ,u 1 dNTPmix ~ ,cc 1 Primer P8 1 ,ul Primer T3 1 ,u 1 3.3x PCR buffer 7.5 ,ul 25 mM MgClz 1.2 ,C.L1 Distilled water 11.8 ,(.cl Total 24.5 ,ul Mineral oil (20 ,(.cl) was overlaid on the aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,ccl, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 40 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1 min. Finally, the extension reaction of fragment was carried out at 72 °C
for 10 min, thus completing PCR. The DNA fragment thus obtained will be referred to as "Fragment D"
hereinafter.
13. DNA Sequencing Direct sequencing was performed on Fragment D by the dye-terminator method, relaying on Primer P8.

14. Formation of a Full-Length DNA
On the basis of the base sequences for Fragments A, B, C, and D, a sense primer, 5'-AGAGCAGCAG AGGTGGCTGC
ACT-3' (the base sequence set forth in SEQ ID NO: 15 in the Sequence Listing) and an antisense primer, 5'-GGCTTGTTCC TCAGCTGGGA CTG-3' (the base sequence set forth in SEQ ID NO: 16 in the Sequence Listing) were designed. Employing this and a human fetus brain cDNA
library as a template, PCR was performed under the following conditions:
CDNA 1 ,Cll dNTPmix 1 ,Ct 1 Sense primer 1 ,ul Antisense primer 1 ,ul 3.3x PCR buffer 7.5 ,u 1 mM MgClz 1.2 ,ul Distilled water 11.8 ,u 1 Total 24.5 ,ul Mineral oil (15 ,c.~l) was overlaid on the aforementioned composition and it was allowed to stand at 96 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,(.cl, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 40 times to allow reaction: at 94 °C for 30 sec, at 60 °C

for 30 sec, and then at 72 °C for 1.5 min. Finally, the extension reaction of fragment was carried out at 72 °C for 10 min, thus completing PCR. This full-length cDNA was sequenced by the dye-terminator method.
It was then ascertained that the objective cDNA
containing the full-length coding region had been obtained. This gene was determined to be a human homologue gene of the neuralized gene of Drosophila.
This base sequence is shown in SEQ ID No. 2 in the Sequence Listing. The amino acid sequence of human neuralized protein that this gene encodes is shown in SEQ ID NO: 1 in the Sequence Listing.

15. Preparation of a Transformant The full-length human neuralized cDNA obtained above was inserted into the BamHI-KpnI site of pBTM116HA: an 83mer linker containing a HA tag had been inserted into the EcoRI-Pst site of a plasmid vector pBTM116HA. This plasmid vector was introduced to E.
coli DH5cr to prepare the transformant.
EXAMPLE 2 Expression of the Neuralized mRNA in Various Tissues (1) Each 2 ,u g of poly(A)+RNA (mRNA) from a variety of human tissues and poly(A)+RNA (mRNA) from a variety of human cells was blotted to a membrane: Human Multiple Tissue Northern Blot I, II and Human Cancer Cell Line Multiple Tissue Northern Blot (available from CLONTECH
Laboratories, Inc.). Fragment A labeled with 32P-CTP
using a Random Primed Labeling kit (Registered Trademark: available from Takara Shuzo Co. Ltd.) was hybridized to the membrane under high stringency conditions by following the description in Molecular Cloning: A Laboratory Manual 2nd ed; p 7.39-7.52. An analysis by northern blot hybridization was conducted.
Results on the respective tissues are shown in Fig.
1. It was understood that the neuralized gene is expressed in brain and muscle tissues.
The results showed no clear expression with respect to any of the cells.
From the above results, it is thought that the neuralized gene and the neuralized protein are nerve-specific molecules.
EXAMPLE 3 Expression of the Neuralized mRNA in Various Tissues (2) Total RNAs were extracted from a normal human cerebral tissue (three samples), astrocytoma tissues (anaplastic astrocytoma: one sample, and glioblastoma:
three samples), and a glioma cell strain by acid-guanidinium thocyanate-phenol-chloroform method. These RNAs were subjected to electrophoresis on an agarose gel, and then they were stained with ethidium-bromide.

The total RNAs were denatured with 6~ formaldehyde-1~
agarose gel and transcribed onto a nylon membrane (Hybond ~: available from Amersham Pharmacia Biotech Inc.).
Hybridization was performed by allowing reaction at 42 ° C overnight in the following buffer:
a -32P-dCTP labeled probe (the full-length human neuralized cDNA) 50 mM HEPES (pH 7) 0.75 M NaCl 50~ Formamide 3.5~ SDS
SxDenhart~s 2mM EDTA
0.1~ SDS
200 ,u g/ml Salmon sperm DNA
After the membrane was washed in the same buffer with 2xSSC and 0.1~ SDS at 55 ° C for 20 min, it was further washed with lxSSC and 0.1~ SDS at 55 ° C for 20 min.
Subsequently, the membrane was exposed by being irradiated with X-ray at -80 ° C for three nights.
The result is shown in Fig. 2. As is apparent from Fig. 2, expression of the human neural-ized mRNA

was noted in the normal human cerebral tissue and the anaplastic astrocytoma, whereas it was not noted in the glioblastoma and the glioma.
EXAMPLE 4 Analysis of Variation of the Human Neuralized in Malignant Astrocytoma 1. Synthesis of cDNA
Total RNAs were extracted from a normal brain tissue, medulloblastoma Med-3, and glioma cell strains (U87MG, U251MG, U138MG, Hs638, TG98, A172, SF126, U373MG, U105MG, NP1, NP2, and RBR17T).
Each 5 ,u g of the total RNAs was incubated in 20 ,u 1 of the following composition at 37 ° C for 1 h and a cDNA was synthesized.
Reaction Solution:
50 mM Tris-HC1 (pH 8.3) 10 mM DTT
10 mM KC1 0.5 mM dNTP
30 ,(,cg RNAsin (available from GIBCO BRL Inc.) 150 ,u g Superscriptll reverse transcriptase (available from GIBCO BRL Inc.) 2. RT-PCR
PCR was performed on 2 ,(,l,g of the reverse transcriptase product at 30 cycles with a Geneamp PCR
- System 9600 (available from Perkin-Elmer Ceutus Inc.), using 25 ,u 1 of rTth DNA polymerase (available from Applied Biosystems Inc.).
A set of the following primers was used as primers for PCR.
neuS-13: 5'-AGCTGGTGCTCCCGGACTGTCTG-3' (the base sequence set forth in SEQ ID NO: 19 in the Sequence Listing) neuAS-stop: 5'-AGCTGGTGCTCCCGGACTGTCTG-3' (the base sequence set forth in SEQ ID NO: 20 in the Sequence Listing) In order to ascertain that the cDNA synthesis had been successfully carried out, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified using the following primers.
GAPDH sense primer: 5'-AAGGCTGGGGCTCATTTGCAG-3' (the base sequence set forth in SEQ ID NO: 21 in the Sequence Listing) GAPDH antisense primer: 5'-CCAAATTCGTTGTCATACCAGG-3' (the base sequence set forth in SEQ ID NO: 22 in the Sequence Listing) The amplified products were subjected to electrophoresis on a 2~ agarose gel.
The result is shown in Fig. 3. As is apparent from the figure, the human neuralized was amplified in the normal human cerebral tissue, the medulloblastoma, and U87MG, whereas it was not amplified in the glioma cells other than U87MG.
EXAMPLE 5 Preparation of Antibodies Recognizing the Neuralized Protein S 1. Preparation of Antigens Peptide A and Peptide B described below were synthesized., Peptide A: a peptide comprising 21 amino acids of from the 123rd Leu to the 143rd Cys of the amino acid sequence of the neuralized protein set forth in SEQ ID
No: 1 in the Sequence Listing.
Peptide B: a peptide comprising an amino acid obtained by adding cysteine (Cys) to the N-terminus of amino acids of from the 272nd Pro to the 291st Asp 15 of the amino acid sequence of the neuralized protein set forth in SEQ ID NO: 1 in the Sequence Listing.
Addition of cysteine was done to effect conjugation with hemocyanin from Macroschisma sinensis (KHL). Iwaki Glass Co. Ltd. was commissioned for their 20 synthesis.
The synthesized peptides (2 mg) were conjugated to 2 mg of maleimido-KLH (available from Pierce Chemical Co.). The reaction was carried out according to the method as described in the manual for a kit from Pierce Chemical Co.
2 . Immunization For each antigen (Peptide A and Peptide B), 100 ,cc 1 of an antigen solution (1 ,C.tg/ml), 0.5 ml of PBS and 0.5 ml of a Freund~s complete adjuvant (available from Difco Inc.) were taken in a syringe and mixed to prepare an emulsion, which was subcutaneously inoculated to a rabbit at four places on its back.
One week later, a second immunization was carried out. The adjuvant was changed to a Freund~s incomplete adjuvant (available from Difco Inc.) from the second time, after which the immunization was done. The other manipulations were the same as those in the first time.
After the second time, immunization was done six times in total at intervals of one week.
3. Purification of Antibodies One week after the final immunization, blood was collected. After this blood was allowed to stand at room temperature for 3 h and sufficient coagulation was effected, centrifugation was carried vut at 3,000 rpm for 5 min to recover the supernatant (serum).
This serum was salted out by the addition of ammonium sulfate to bring the final concentration to 50~. This sample was centrifuged and fractions containing an antibody were subjected to precipitation.
Subsequently, the precipitate was dissolved in PBS and was further dialyzed against PBS.
Then, the antibody was purified by affinity chromatography, using a Protein G Sepharose column (Registered Trademark: available from Pharmacia Inc.).
As a result, a total amount of 370 mg of IgG fractions was obtained.
The IgG fractions were purified by affinity chromatography, using a prepared column: the column was prepared by binding the peptide used in immunization to NHS-activated Sepharose (available from Pharmacia Inc.) according to its attached manual. As a result, a total amount of 5 mg of a purified antibody was obtained.
4. Determination of Antibody Titers The titers of the resultant antibodies were measured by ELISA.
(1) Each of the antigen preparations (Peptide A
and Peptide B) was diluted with PBS to give a concentration of 25 ,u g/ml, and it was fractionally poured to each well of a 96-well ELISA plate (XENOBIND
~: available from XENOPORE Corp.) at 50,Ct1, which was allowed to stand at 4 ° C overnight.
(2) The antigen preparation was discarded and BLOCKAGE (Registered Trademark: available from Dainippon Pharmaceuticals Co. Ltd.) diluted fourfold with distilled water was fractionally poured to each well at 200 ,C.~1. Blocking was carried out by allowing it to stand at room temperature for 2 h.
(3) The blocking solution was discarded and the purified antibody was added as a primary antibody. A
serially (in two-fold) PBS-diluted solution of the purified antibody was fractionally poured to each well at 50 ,u 1 so that its concentration could successively S reach 10 ,(.~g/ml, 5 ,(.Lg/ml, 2.5 ,(.Lg/ml, etc. from the first row to the 12th row of the 96 wells. The concentration of the antibody in the 12th row proved to be about 0.5 ,u g/ml. In addition, IgG purified from the blood of a rabbit that was not immunized was used as control. After each antibody was fractionally poured, reaction was allowed to proceed at room temperature for 1 h.
(4) The antibody solution was discarded and the plate was washed with 0.05 Tween20/PBS four times.
Next, a biotinylated rabbit IgG antibody (available from Vector Laboratories, Inc.) that had been 1000-fold diluted with PBS, which served as a secondary antibody solution, was fractionally poured to each well at 50 ,u 1.
Subsequently, reaction was allowed to proceed at room temperature for 30 min.
(5) After the secondary antibody was discarded, the plate was washed with 0.05 Tween20/PBS four times.
An ABC solution diluted 1000-fold (available from Vector Laboratories, Inc.) was fractionally poured to each well at 50 ,(.C1 , and then it was allowed to stand at room temperature for 30 min.

(6) After the ABC solution was discarded, the plate was washed with 0.05$ Tween20/PBS four times.
OPD (orthophenylenediamine)-Hz02/PBS was fractionally poured to each well at 100 ,u 1. After the reaction was quenched with 2N sulfuric acid upon sufficient coloring, absorbance at 490 nm was measured with a microplate reader (available from Bio-Rad Inc.).
From this result, the antibody obtained by immunization of Peptide A (which hereinafter may be referred to as "Antibody A") and the antibody obtained by immunization of Peptide B (which hereinafter may be referred to as "Antibody B") were both higher in their titer compared to the control.
EXAMPLE 6 Western Blotting (1) The coding region of human neuralized cDNA was inserted into the BamHI-KpnI site of pBTM116HA: an 83mer linker containing a HA tag had been inserted into the EcoRI-Pst site of the plasmid vector pBTM116HA.
This plasmid vector was introduced to COS cells.
(2) The COS cells to which the neuralized cDNA had been introduced, the cells of its parent strain (cells to which no neuralized cDNA had been introduced), a human brain tissue, and a human muscle tissue were subjected to dissolution after recovery of the cells and tissues. 1x106 cells were used and a Human Brain Protein Medley (available from CLONTECH Laboratories Inc.) was used for the brain tissue. As a cell lysate, 68 mM Tris-HC1, 14% glycerol, 3% SDS, 0.1 M DTT, 10 ,u g/ml soybean trypsin inhibitor, 1 ,ctg/ml aprotinin, 1 mM
phenyl methyl sulfonyl fluoride (PMSF), and 1 ,C.~g/ml leupeptin were used. The cell lysate (100 ,ccl) was added to a cell pellet, and it was dissolved with thorough stirring. After boiling for 10 min, centrifugation was carried out at 15,000 rpm for 10 min and the supernatant was recovered.
(3) Ten microliters of the supernatant was subjected to electrophoresis on a 10-20% gradient gel.
(4) After electrophoresis, proteins on the gel were transferred onto nitrocellulose membrane using a Trans Blot System (available from Marisol Inc.).
(5) The nitrocellulose membrane was immersed in 10% skim milk/PBS and 0.1% Tween20 and allowed to stand for 1 h to effect blocking. Subsequently, it was washed with 0.3% Tween20/PBS for 5 min twice.
(6) After each of the anti-neuralized antibodies prepared in Example 3 was diluted with PBS to give a concentration of 1 ,c.cg/ml, it was added to the nitrocellulose membrane and allowed to react at room temperature for 40 min. Subsequently, washing was done with 0.3% Tween20/PBS for 5 min three times.
(7) Next, a peroxidase-labeled anti-rabbit IgG

(available from Amersham Pharmacia Biotech Inc.) was diluted with PBS 20000-fold and it was added to the nitrocellulose membrane and further allowed to react at room temperature for 40 min. Subsequently, washing was done with 0.3~ Tween20/PBS for 5 min three times.
(8) Next, only 5 ml of the coloring solution from an EC1 kit (available from Amersham Pharmacia Biotech Inc.) was added to the nitrocellulose membrane and it was allowed to react for 1 min. Then, this membrane was exposed to an X-ray film for 20 sec and the film was developed, after which a photograph was taken.
From this result, when either of the antibodies was used, a band for the neuralized protein was detected in any of the cell lysates of the COS cells, the brain tissue, and the muscle tissue. This ascertained that Antibody A and Antibody B both recognize the neuralized protein.
EXAMPLE 7 Tissue Staining Employing Antibody A and Antibody B, immunostaining was conducted on a tissue of human nervous system and a human brain tissue, according to the following manipulations.
(1) The human brain tissue was put in a compound and a frozen block was prepared. The block was made into-a frozen section, using a cryostat, and the section was placed on a slide glass.
(2) Next, the frozen tissue section was subjected to 10~ buffered formalin treatment for 10 min to effect fixation. Then, washing was done with PBS (pH 7.5) for 5 min three times. Further, 0.3% Hz02/methanol treatment was done for 30 min and washing was done with PBS twice.
(3) Over the tissue on the slide glass was placed 0.15% donkey normal serum/PBS and blocking was conducted by allowing it to stand at room temperature for 1 h.
(4) Antibodies A and B diluted with PBS (to give a concentration of 1 ,ccg/ml) were added to the tissues on the slide glasses, respectively. After allowing them to react at room temperature for 1 h, the slide glasses were washed with PBS (pH 7.5) for 5 min three times.
(5) A biotinylated rabbit IgG antibody (available from Amersham Pharmacia Biotech Inc.) diluted with PBS
1000-fold was added to the tissues. They were allowed to react at room temperature for 1 h. Then, washing was done with PBS (pH 7.5) for 5 min three times.
After addition of a peroxidase-labeled avidin-biotin complex solution and reaction for 40 min, washing was done with PBS for 5 min three times.
(6) The slide glasses were immersed in 0.1~
diaminobenzidine (DAB) and 0.02 HzOz/PBS (pH 7.5) solutions and allowed to react at room temperature for min. Then, the slide glasses were transferred into distilled water to quench the reaction. After staining with hematoxylin, washing was done with running water.
5 When either of the antibodies was used, staining of the neuralized protein in the brain tissue was observed.
EXAMPLE 8 Isolation of the Murine Neuralized cDNA and 10 Determination of Its Base Sequence and the Amino Acid Sequence Encoded by Said Gene 1. Obtaining of DNA Fragments A murine neonatal brain cDNA library was used as a cDNA library and PCR was performed under the following conditions.
Employing the aforementioned Primer P3 in view of the base sequence of Fragment A and the murine newborn offspring brain cDNA library as a template, single PCR
was performed under the following conditions:
Murine new born offspring Brain cDNA library (l,ccg/ml) 1 ,ctl dNTPmix 0.6 ,C.ll Primer P3 1 ,u l 3.3x PCR buffer 4.5 ,ul 25 mM MgCl2 0.72 ,ul Distilled water 6.68 ,C.~1 Total 14 . 5 ,u 1 Mineral oil (15 ,(.C1) was overlaid on the S aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,C.tl, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 50 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1.5 min.
Subsequently, the extension reaction of fragment was carried out at 55 °C for 2 min and finally at 72 °C for 10 min, thus completing PCR.
This PCR product was used as a template and second PCR was performed. Employing the aforementioned Primers P4 and T3, PCR was performed under the following conditions:
PCR product 2 ,C11 dNTPmix 1 ,u 1 Primer P4 1 ,CL 1 Primer T3 1 ,cc 1 3.3x PCR buffer 7.5 ,ul 2 5 mM MgC 12 1 . 2 ,Ct l Distilled water 11.8 ,c..~l Total 24.5 ,ul Mineral oil (20 ,u 1) was overlaid on the aforementioned composition and it was allowed to stand at 94 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,u 1, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 40 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1 min. Subsequently, the extension reaction of fragment was carried out at 55 °C for 2 min and finally at 72 °C for 10 min, thus completing PCR. The DNA fragment will be referred to as "Fragment MA" hereinafter.
Fragment MA that had been obtained through the 5'-extension reaction as described above was subjected to electrophoresis on a minigel (0.75% agarose gel). A
band for Fragment MA was sliced out. Fragment MA was recovered with a GeneClean (available from BIO 101, Inc.) and was examined for the presence of its band on minigel electrophoresis.
Fragment MA (1 ,CCl) was taken and diluted with 99 ,ul of TE. Absorbance at 260 nm (A260) was measured and DNA concentrations were computed: when A260 was 1.0, the DNA concentration was taken as 50 ,(.tl/ml. Fragment MA was diluted with TE so as to give a DNA
concentration of 1 ,u 1/ml.
This diluted solution was subjected to DNA
sequencing according to the dye-terminator method, using an autosequencer (ABI Model 373A) and the base sequence of Fragment MA was determined.
2. Obtaining of the Murine Neuralized cDNA
Sense Primer P9, 5'-GACTCCATCG GGGGCTCCTT CCC-3' (the base sequence set forth in SEQ ID NO: 17 in the Sequence Listing), which served as a 5'-primer and which corresponded to a part of Fragment MA, antisense Primer P10, 5'-CTAGGAGCTG CGGTAGGTCT TGA-3' (the base sequence set forth in SEQ ID No. 18 in the Sequence Listing), which served as a 3'-primer, were synthesized with a DNA synthesizer (ABI Model 392).
Employing the aforementioned primers as a template and the murine neonatal brain cDNA library (available from CLONTECH Laboratories Inc.), PCR was performed under the following conditions:
CDNA 1 ,LC 1 dNTPmix 1 ,u l Sense primer 1 ,ul Antisense primer 1 ,ul 3.3x PCR buffer 7.5 ,ul mM MgClz 1.2 ,ccl Distilled water 11.8 ,ul Total 24.5 ,u1 Mineral oil (15 ,C.cl) was overlaid on the aforementioned composition and it was allowed to stand at 96 °C for 5 min. Then, Taq polymeraseXL, 0.5 ,u 1, (Registered Trademark: available from Takara Shuzo Co.
Ltd.) was added. The following cycle was repeated 40 times to allow reaction: at 94 °C for 30 sec, at 60 °C
for 30 sec, and then at 72 °C for 1.5 min. Finally, the extension reaction of fragment was carried out at 72 °C for 10 min, thus completing PCR.
This full-length cDNA was sequenced according to the dye-terminotor method. This gene was determined to be a murine homologous gene of the neuralized gene of Drosophila. This base sequence is shown in SEQ ID NO:
4 in the Sequence Listing. In addition, the amino acid sequence of the murine neuralized protein that this gene encodes is shown in SEQ ID NO: 3 in the Sequence Listing.
3. Preparation of a Transformant A murine neuralized gene comprising the aforementioned base sequence was inserted into the TA
cloning site of a pCRII vector (Registered Trademark, available from Invitrogen Corp.), and said vector was introduced to E. coli DH5a to prepare a transformant.
INDUSTRIAL APPLICABILITY
The neuralized gene of Drosophila is one of the gene groups that function during the formation of nerve cells. Since its deficiency causes the abnormal proliferation of immature nerve cells at a larva stage of Drosophila, it is believed to be the gene regulating proliferation, differentiation-inducing signals of cells in the nervous system. This invention has revealed that the neuralized gene, which has traditionally been known in Drosophila, exists in vertebrate animals, more specifically in mammals such as human and mouse. The human neuralized gene appears in brain and muscle tissues with high frequency and is a potential candidate for one of the genes that participate in the development of malignant tumors.
The neuralized proteins, the genes encoding said proteins, the antisense genes of said genes, and the antibodies recognizing said proteins according to this invention are powerful tools as a reagent for analyzing cerebral tumor.

SEQUENCE
LISTING

<110> SUMOTOMO ELECTRICINDUSTRIES,LTD

<120> NEURALIZED NS,POLYNUCLEOTIDESCODING INS, PROTEI THE
PROTE

AND ANTIBODIES RE COGNIZING NS
THE
PROTEI

<130> SEI98-31PCT

<150> JP 9-313211 <151> 1997-11-14 <160> 23 <170> PatentIn Ver.
2.0 <210> 1 <211> 574 <212> PRT

<213> Homo sapiens <400> 1 Met Gly Asn Asn SerIle Ser Pro Gly Pro Phe Ser Pro Leu Arg Asn Ser Arg Ala Pro HisPro Asn Lys Ser Gly Arg Gly Gln Leu Asp Ile Gly Pro Phe Pro SerHis Cys His Gln His Val Thr Arg His Lys Lys Cys Pro Ala Val SerGly Leu Ala Pro Leu Leu Pro Gly Pro Thr Leu Phe His Pro His GlySer Ile Met Leu His Thr Lys Gln Leu Asp Ser Lys Ala Val Lys AlaSer Cys Ala Thr Ser Arg Gln Phe Asn Ile Phe Asn Arg Pro Val TyrGlu Val Leu Ile Lys Leu Ile Gln Arg Lys Thr Lys Gln Cys Cys Trp Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys Asp Pro Ser Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys Gly Arg Val Phe His Arg Ile Asn Asp Ser Ala Val Met Leu Phe Phe Ser Gly Val Arg Thr Ala Asp Pro Leu Trp Ala Leu Val Asp Val Tyr Gly Leu Thr Arg Gly Val Gln Leu Leu Asp Ser Glu Leu Val Leu Pro Asp Cys Leu Arg Pro Arg Ser Phe Thr Ala Leu Arg Arg Pro Ser Leu Arg Arg Glu Ala Asp Asp Ala Arg Leu Ser Val Ser Leu Cys Asp Leu Asn Val Pro Gly Ala Asp Gly Asp Glu Ala Ala Pro Ala Ala Gly Cys Pro Ile Pro Gln Asn Ser Leu Asn Ser Gln His Ser Arg Ala Leu Pro Ala Gln Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg Ala Gly Ala His Val Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Val Glu His Gly Arg Asp Glu Arg Ala Leu Val Phe Thr Ser Arg Pro Val Arg Val Ala Glu Thr Ile Phe Val Lys Val Thr Arg Ser Gly Gly Ala Arg Pro Gly Ala Leu Ser Phe Gly Val Thr Thr Cys Asp Pro Gly Thr Leu Arg Pro Ala Asp Leu Pro Phe Ser Pro Glu Ala Leu Val Asp Arg Lys Glu Phe Trp Ala Val Cys Arg Val Pro Gly Pro Leu His Ser Gly Asp Ile Leu Gly Leu Val Val Asn Ala Asp Gly Glu Leu His Leu Ser His Asn Gly Ala Ala Ala Gly Met Gln Leu Cys Val Asp ~Ala Ser Gln Pro Leu Trp Met Leu Phe Gly Leu His Gly Thr Ile Thr Gln Ile Arg Ile Leu Gly Ser Thr Ile Leu Ala Glu Arg Gly Ile Pro Ser Leu Pro Cys Ser Pro Ala Ser Thr Pro Thr Ser Pro Ser Ala Leu Gly Ser Arg Leu Ser Asp Pro Leu Leu Ser Thr Cys Ser Ser Gly Pro Leu Gly Ser Ser Ala Gly Gly Thr Ala Pro Asn Ser Pro Val Ser Leu Pro Glu Ser Pro Val Thr Pro Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys Tyr Glu His Ala Val Asp Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys Tyr Ala Cys Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys Cys Pro Ile Cys Arg Arg Pro Ile Lys Asp Ile Ile Lys Thr Tyr Arg Ser Ser <210> 2 <211> 2207 <212> DNA
<213> Homo sapiens <221> CDS

<222> (411)..(2135) <400> 2 ccgaacgccc acgccagcga ccctgactct atggccccgg gggagcgcgc cggagccgcc 60 gcgccgccca cccccagccg gaaccctagc gtcccgggga gcaagcgggg 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 Met Gly aac aac ttc tcc agt atc ccc tcg ctg ccc cga gga aac ccg agc cgc 464 Asn Asn Phe Ser Ser Ile Pro Ser Leu Pro Arg Gly Asn Pro Ser Arg gcg ccg cgg ggc cac ccc cag aac ctc aaa gac tct atc ggg ggc ccc 512 Ala Pro Arg Gly His Pro Gln Asn Leu Lys Asp Ser Ile Gly Gly Pro ttc ccc gtc act tct cac cga tgc cac cac aag cag aag cac tgt ccg 560 Phe Pro Val Thr Ser His Arg Cys His His Lys Gln Lys His Cys Pro gca gtg ctg ccc agc ggg ggg ctc cca gcc acg ccg ctg ctc ttc cac 608 Ala Val Leu Pro Ser Gly Gly Leu Pro Ala Thr Pro Leu Leu Phe His ccg cac acc aag ggc tcc cag atc ctc atg gac ctc agc cac aag get 656 Pro His Thr Lys Gly Ser Gln Ile Leu Met Asp Leu Ser His Lys Ala gtc aag agg cag gcc agc ttc tgc aac gcc atc acc ttc agc aac cgc 704 Val Lys Arg Gln Ala Ser Phe Cys Asn Ala Ile Thr Phe Ser Asn Arg ccg gtc ctc atc tac gag caa gtc agg ctg aag atc acc aag aag cag 752 Pro Val Leu Ile Tyr Glu Gln Val Arg Leu Lys Ile Thr Lys Lys Gln tgc tgc tgg agc ggg gcc ctg cgg ctg ggc ttc acc agc aag gac ccg 800 Cys Cys Trp Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys Asp Pro tcc cgc atc cac cct gac tcg ctg ccc aag tac gcc tgc ccc gac ctg 848 Ser Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro Asp Leu gtg tcc cag agt ggc ttc tgg gcc a,ag gcg ctg cct gag gag ttt gcc 896 Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu Phe Ala aat gag ggc aac atc atc gca ttc tgg gtg gac a,ag a,ag ggc cgt gtc 944 Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys Gly Arg Val ttc cac cgc atc a,ac gac tcg get gtt atg ctg ttc ttc agc ggg gtc 992 Phe His Arg Ile Asn Asp Ser Ala Val Met Leu Phe Phe Ser Gly Val cgc acg gcc gac ccg ctc tgg gcc ctg gtg gac gtc tac ggc ctc acg 1040 Arg Thr Ala Asp Pro Leu Trp Ala Leu Val Asp Val Tyr Gly Leu Thr cgg ggc gtc cag ctg ctt gat agc gag ctg gtg ctc ccg gac tgt ctg 1088 Arg Gly Val Gln Leu Leu Asp Ser Glu Leu Val Leu Pro Asp Cys Leu cgg ccg cgc tcc ttc acc gcc ctg cgg cgg ccg tcg ctg cgg cgc gag 1136 Arg Pro Arg Ser Phe Thr Ala Leu Arg Arg Pro Ser Leu Arg Arg Glu gcg gac gac gcg cgc ctc tcg gtg agc cta tgc gac ctc aac gtg ccg 1184 Ala Asp Asp Ala Arg Leu Ser Val Ser Leu Cys Asp Leu Asn Val Pro ggc gcg gac ggc gac gag gcc gcg ccg gcc gcc ggc tgc ccc atc ccg 1232 Gly Ala Asp Gly Asp Glu Ala Ala Pro Ala Ala Gly Cys Pro Ile Pro cag a,ac tca ctc aac tcg cag cac agc cgc gcg ctg ccg gcg cag ctc 1280 Gln Asn Ser Leu Asn Ser Gln His Ser Arg Ala Leu Pro Ala Gln Leu gac ggc gac ctg cgt ttc cac gcc ctg cgc gcc ggc gcg cac gtc cgc 1328 Asp Gly Asp Leu Arg Phe His Ala Leu Arg Ala Gly Ala His Val Arg atc ctc gac gag cag acg gtg gcg cgc gtg gag cac ggg cgc gac gag 1376 Ile Leu Asp Glu Gln Thr Val Ala Arg Val Glu His Gly Arg Asp Glu cgc gcg ctc gtc ttc acc agc cgg ccc gtg cgc gtg gcc gag acc atc 1424 Arg Ala Leu Val Phe Thr Ser Arg Pro Val Arg Val Ala Glu Thr Ile ttc gtc a,ag gtc acg cgc tcg ggt ggc gcg cgg ccc ggc gcg ctg tcg 1472 Phe Val Lys Val Thr Arg Ser Gly Gly Ala Arg Pro Gly Ala Leu Ser ttc ggc gtc acc acg tgc gac ccc ggc acg ctg cgg ccg gcc gac ctg 1520 Phe Gly Val Thr Thr Cys Asp Pro Gly Thr Leu Arg Pro Ala Asp Leu cct ttc agc cct gag gcc ctg gtg gac cgc aag ga,a, ttc tgg gcc gtg 1568 Pro Phe Ser Pro Glu Ala Leu Val Asp Arg Lys Glu Phe Trp Ala Val tgc cgc gtg ccc ggg ccc ctg cac agc ggc ga,c atc ctg ggc ctg gtg 1616 Cys Arg Val Pro Gly Pro Leu His Ser Gly Asp Ile Leu Gly Leu Val gtc aa.c gcc gac ggc gag ctg cac ctc agc cac aat ggc gcg gcc gcc 1664 Val Asn Ala Asp Gly Glu Leu His Leu Ser His Asn Gly Ala Ala Ala ggc atg cag ctg tgc gtg gac gcc tcg cag ccg ctt tgg atg ctc ttc 1712 Gly Met Gln Leu Cys Val Asp Ala Ser Gln Pro Leu Trp Met Leu Phe ggc ctg cac ggg acc atc acg cag atc cgc atc ctc ggc tcc act atc 1760 Gly Leu His Gly Thr Ile Thr Gln Ile Arg Ile Leu Gly Ser Thr Ile ctg gcc gag cgg ggt atc ccg tca ctc ccc tgc tcc cct gcc tcc acg 1808 Leu Ala Glu Arg Gly Ile Pro Ser Leu Pro Cys Ser Pro Ala Ser Thr cca acc tcg ccc agt gcC ctg ggc agc cgc ctg tct gac ccc ttg ctc 1856 Pro Thr Ser Pro Ser Ala Leu Gly Ser Arg Leu Ser Asp Pro Leu Leu agc acg tgc agc tct ggc cct ctg ggt agc tct get ggt ggg aca gcc 1904 Ser Thr Cys Ser Ser Gly Pro Leu Gly Ser Ser Ala Gly Gly Thr Ala ccc aat tcg cca gtg agc ctg ccc gag tcg cca gtg acc cca ggt ctg 1952 Pro Asn Ser Pro Val Ser Leu Pro Glu Ser Pro Val Thr Pro Gly Leu ggc cag tgg agc gat gag tgc acc att tgc tat gaa cac gcg gtg gac 2000 Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys Tyr Glu His Ala Val Asp acg gtc atc tac aca tgt ggc cac atg tgc ctc tgc tac gcc tgt ggc 2048 Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys Tyr Ala Cys Gly ctg cgc ctc aag aag get ctg cac gcc tgc tgc ccc atc tgc cgc cgc 2096 Leu Arg Leu Lys Lys Ala Leu His Ala Cys Cys Pro Ile Cys Arg Arg ccc atc aag gac atc atc aag acc tac cgc agc tcc tag cccgttgcgg 2145 Pro Ile Lys Asp Ile Ile Lys Thr Tyr Arg Ser Ser tggcccatcc cgcataccca tcttctcggg cttcagccca gtcccagctg aggaacaagc 2205 ca 2207 <210> 3 <211> 546 <212> PRT
<213> Mus <400> 3 Asp Ser Ile Gly Gly Ser Phe Pro Val Pro Ser His Arg Cys His His Lys Gln Lys His Cys Pro Pro Thr Leu Ser Gly Gly Gly Leu Pro Ala Thr Pro Leu Leu Phe His Pro His Thr Lys Gly Ser Gln Ile Leu Met Asp Leu Ser His Lys Ala Val Lys Arg Gln Ala Ser Phe Cys Asn Ala Ile Thr Phe Ser Asn Arg Pro Val Leu Ile Tyr Glu Gln Val Arg Leu Lys Xa.a, Thr Lys Lys Gln Cys Cys Trp Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys Asp Pro Ser Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys Gly Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met Leu Phe Phe Ser Gly Val Arg Thr Val Asp Pro Leu Trp Ala Leu Val Asp Val Tyr Gly Leu Thr Arg Gly Val Gln Leu Leu Asp Ser Glu Leu Val Leu Pro Asp Cys Leu Arg Pro Arg Ser Phe Thr Ala Leu Arg Arg Pro Ser Leu Arg Cys Glu Ala Asp Glu Ala Arg Leu Ser Val Ser Leu Cys Asp Leu Asn Val Pro Gly Ala Asp Gly Asp Asp Gly Ala Pro Pro Ala Gly Cys Pro Ile Pro Gln Asn Ser Leu Asn Ser Gln His Ser Arg Ala Leu Pro Ala Gln Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg Ala Gly Ala His Val Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Leu Glu His Gly Arg Asp Glu Arg Ala Leu Val Phe Thr Ser Arg Pro Val Ser Val Ala Glu Thr Ile Phe Ile Lys Val Thr Arg Ser Gly Gly Gly Arg Glu Gly Ala Leu Ser Phe Gly Val Thr Thr Cys Asp Pro Gly Thr Leu Arg Pro Ala Asp Leu Pro Phe Ser Pro Glu Ala Leu Val Asp Arg Lys Glu Phe Trp Ala Val Cys Arg Val Pro Gly Pro Leu His Ser Gly Asp Ile Leu Gly Leu Val Val Asn Ala Asp Gly Glu Leu His Leu Ser His Asn Gly Ala Ala Ala Gly Met Gln Leu Cys Val Asp Ala Ser Gln Pro Leu Trp Met Leu Phe Ser Leu His Gly Ala Ile Thr Gln Val Arg Ile Leu Gly Ser Thr Ile Met Thr Glu Arg Gly Gly Pro Ser Leu Pro Cys Ser Pro Ala Ser Thr Pro Thr Ser Pro Ser Ala Leu Gly Ile Arg Leu Ser Asp Pro Leu Leu Ser Thr Cys Gly Ser Gly Pro Leu Gly Gly Ser Ala Gly Gly Thr Ala Pro Asn Ser Pro Val Ser Leu Pro Glu Pro Pro Val Thr Pro Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys Tyr Glu His Ala Val Asp Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys Tyr Ser Cys Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys Cys Pro Ile Cys Arg Arg Pro Ile Lys Asp Ile Ile Lys Thr Tyr Arg _ 530 535 540 Ser Ser <210> 4 <211> 1641 <212> DNA
<213> Mus <221> CDS
<222> (1)..(1641) <400> 4 gac tcc atc ggg ggc tcc ttc ccg gtg ccc tct cac cga tgc cat cac 48 Asp Ser Ile Gly Gly Ser Phe Pro Val Pro Ser His Arg Cys His His aag cag aag cat tgc ccg cct acg ctg tca ggt ggg ggg ctc ccg gcc 96 Lys Gln Lys His Cys Pro Pro Thr Leu Ser Gly Gly Gly Leu Pro Ala acg ccg ctg ctc ttc cac ccc cac act aag ggc tcc cag atc ctc atg 144 Thr Pro Leu Leu Phe His Pro His Thr Lys Gly Ser Gln Ile Leu Met gac ctc agc cac aag gcc gtc aag agg cag gcc agc ttc tgc aat gcc 192 Asp Leu Ser His Lys Ala Val Lys Arg Gln Ala Ser Phe Cys Asn Ala atc acc ttc agt aac cgc ccg gtg ctc atc tac gag caa gtc agg ctg 240 Ile Thr Phe Ser Asn Arg Pro Val Leu Ile Tyr Glu Gln Val Arg Leu 65 70 ~ 75 80 aag ntc acc aag aag caa tgc tgc tgg agc ggg gcc ctg cga ctt ggc 288 Lys Xaa Thr Lys Lys Gln Cys Cys Trp Ser Gly Ala Leu Arg Leu Gly ttc acc agc aag gac cct tcc cgc atc cac ccc gac tcg ctg ccc aag 336 Phe Thr Ser Lys Asp Pro Ser Arg Ile His Pro Asp Ser Leu Pro Lys tac gcc tgc cct gac ctg gtg tct cag agt ggc ttc tgg gcc aaa gca 384 Tyr Ala Cys Pro Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala ttg cct gag gag ttt gcc aac gag ggc aac atc att gcc ttc tgg gtg 432 Leu Pro Glu Glu Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val gac aag aag ggc cgc gtc ttc tac cgg atc aat gag tca get get atg 480 Asp Lys Lys Gly Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met ctt ttc ttc agt ggg gtc cgg acg gtg gac ccg ctc tgg gcc ctg gtg 528 Leu Phe Phe Ser Gly Val Arg Thr Val Asp Pro Leu Trp Ala Leu Val gac gtc tac ggc ctc acg cgg ggt gtc cag ctg cta gac agc gag ctg 576 Asp Val Tyr Gly Leu Thr Arg Gly Val Gln Leu Leu Asp Ser Glu Leu gtg ctg ccc gac tgc ctg cgg ccg cgc tcc ttc acc gcg ctg cgg cgg 624 Val Leu Pro Asp Cys Leu Arg Pro Arg Ser Phe Thr Ala Leu Arg Arg ccg tcg ctg cgg tgc gag gcg gat gaa gcg cgc ctg tcg gtg agc ctg 672 Pro Ser Leu Arg Cys Glu Ala Asp Glu Ala Arg Leu Ser Val Ser Leu tgc gac ctc aac gtg ccg gga gcc gac ggc gac gac ggc gca ccg cct 720 Cys Asp Leu Asn Val Pro Gly Ala Asp Gly Asp Asp Gly Ala Pro Pro gcc ggc tgc ccg atc ccg cag aac tcg ctc aat tct cag cac agc cgc 768 Ala Gly Cys Pro Ile Pro Gln Asn Ser Leu Asn Ser Gln His Ser Arg gcg ctg ccg gcg cag ctc gac ggc gac ctg cgc ttc cac gcg ctt cgc 816 Ala Leu Pro Ala Gln Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg gcc ggc gcg cac gtc cgc atc ctg gac gag cag acg gtg gcg cgc ctg 864 Ala Gly Ala His Val Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Leu gag cac ggg cgc gac gag cgc gcg ctc gtc ttc acc agc cgg cct gtg 912 Glu His Gly Arg Asp Glu Arg Ala Leu Val Phe Thr Ser Arg Pro Val agc gtg gcc gag acc atc ttc atc aag gtc acg cgc tcg ggc ggg ggg 960 Ser Val Ala Glu Thr Ile Phe Ile Lys Val Thr Arg Ser Gly Gly Gly cga gcg ggc gcg ctg tcc ttc ggg gtc acc acg tgt gac cct ggc acg 1008 Arg Ala Gly Ala Leu Ser Phe Gly Val Thr Thr Cys Asp Pro Gly Thr ctg cgg ccc gcg gac ctg ccc ttc agc ccc gag gcc ctg gtg gac cgc 1056 Leu Arg Pro Ala Asp Leu Pro Phe Ser Pro Glu Ala Leu Val Asp Arg aag gag ttc tgg gcg gtg tgt cgc gtg ccc ggg cct ctg cac agc ggc 1104 Lys Glu Phe Trp Ala Val Cys Arg Val Pro Gly Pro Leu His Ser Gly gac atc ctg ggc ctg gtg gtc aac gcg gac gga gag ctg cac ctg agt 1152 Asp Ile Leu Gly Leu Val Val Asn Ala Asp Gly Glu Leu His Leu Ser cac aac ggc gcg gcg gcc ggc atg cag ctg tgc gtg gat gcc tcg cag 1200 His Asn Gly Ala Ala Ala Gly Met Gln Leu Cys Val Asp Ala Ser Gln ccc ctc tgg atg ctc ttc agc ctg cat ggc gcc atc acg cag gtc cgc 1248 Pro Leu Trp Met Leu Phe Ser Leu His Gly Ala Ile Thr Gln Val Arg atc ctc ggc tcc acc atc atg act gaa cgg ggt ggc cca tct ctc ccc 1296 Ile Leu Gly Ser Thr Ile Met Thr Glu Arg Gly Gly Pro Ser Leu Pro tgc tca cct gcc tcc act cca acc tca ccc agt gcc ctg ggc atc cgc 1344 Cys Ser Pro Ala Ser Thr Pro Thr Ser Pro Ser Ala Leu Gly Ile Arg ctc tct gac ccc ctg ctc agc acc tgc ggt tct ggg ccc cta ggt ggc 1392 Leu Ser Asp Pro Leu Leu Ser Thr Cys Gly Ser Gly Pro Leu Gly Gly tct get gga ggg aca gcc ccc aac tca cct gtg agc ctg ccc gag cca 1440 Ser Ala Gly Gly Thr Ala Pro Asn Ser Pro Val Ser Leu Pro Glu Pro ccg gtg acc cca ggt ctg ggc cag tgg agt gat gaa tgc acc att tgc 1488 Pro Val Thr Pro Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys tat gaa cac gca gtg gat aca gtc atc tac acg tgt ggc cac atg tgc 1536 Tyr Glu His Ala Val Asp Thr Val Ile Tyr Thr Cys Gly His Met Cys ctg tgc tac tcc tgt ggc ctg cgc ctc a,ag aag 1584 gcc ctg cac gcc tgc Leu Cys Tyr Ser Cys Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys tgc ccc atc tgc cgt cgc ccc atc aag gac atc atc 1632 aag acc tac cgc Cys Pro Ile Cys Arg Arg Pro Ile Lys Asp Ile Ile Lys Thr Tyr Arg agc tcc tag 1641 Ser Ser <210> 5 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 5 tagacgtcca ccagggccca gac 23 <210> 6 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 6 cggagcactc tccacgactc tat 23 <210> 7 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 7 agcactgccg gacagtgctt ctg 23 <210> 8 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 8 gtggtggcat cggtgagaag tga 23 <210> 9 <211> 17 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 9 attaaccctc actaaag 17 <210> 10 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 10 ggccgtgtct tccaccgcat caa 23 <210> 11 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 11 ctcggctgtt atgctgttct tca 23 <210> 12 <211> 17 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 12 aatacgactc actatag 17 <210> 13 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 13 gtggacgcct cgcagccgct ttg 23 <210> 14 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 14 cgatgagtgc accatttgct atg 23 <210> 15 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 15 agagcagcag aggtggctgc act 23 <210> 16 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 16 ggcttgttcc tcagctggga ctg 23 <210> 17 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 17 gactccatcg ggggctcctt ccc 23 <210> 18 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 18 ctaggagctg cggtaggtct tga 23 <210> 19 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 19 agctggtgct cccggactgt ctg 23 <210> 20 <211> 23 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 20 agctggtgct cccggactgt ctg 23 <210> 21 <211> 21 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 21 aaggctgggg ctcatttgca g 21 <210> 22 <211> 22 <212> DNA

<213> Artificial Sequence <220>

<223> Primer <400> 22 ccaaattcgt tgtcatacca gg 22 <210> 23 <211> 5??

<212> DNA

<213> Home sapiens <400> 23 cagctggacg ccccgntgna ggccgtagac gcccagagcg ggtcggccgt gtccaccagg 60 gcggacccgc tgaagaacag cataacagcc tgcggtggaa gacacggccc gagtcgttga 120 ttcttgtcca cccagaatgc gatgatgttg caaactcctc aggcagcgct ccctcattgc 180 ntggcccaga agccactctg ggacaccagg ttcggggcag gcgtacttgg gcagcgagtc 240 agggtggatg cgggacgggt ccttgctggt gaagccagcc gcaggccccg ctccagcact 300 gcttcttggt gatcttcagc ctgacttgct cgtagatgag gaccgggcgg ttgctgaagg 360 tgatgncgtt gcagaagctg gcctgcctct tgnacagcct tgtggctgag gtccatgagg 420 atctgggagc ccttggtgtg cgggtggaag agcagcggcg tggctgggag cccccgctgg 480 gcagcactgc cggacagtgc ttctgcttgt ggtggcatcg gtgagaagtg acggggaagg 540 ggcccccgat agagtcgtgg agagtgctcc gggtgat 577

Claims (12)

1. A protein comprising an amino acid sequence having at least the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing.

2. A protein comprising an amino acid sequence having at least the amino acid sequence set forth in 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 of the amino acid sequence set forth 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 of the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing.

5. A polynucleotide encoding the protein according to any of claims 1 - 4.

6. A polynucleotide being a part of the polynucleotide according to claim 5 and comprising not less than 12 consecutive bases.

7. A polynucleotide being an antisense polynucleotide or a part of a derivative thereof and comprising not less than 12 consecutive bases, the antisense polynucleotide comprising the base sequence of an antisense strand of the polynucleotide according to claim 5.

8. The polynucleotide according to either claim 5 or claim 7, wherein the polynucleotide is chemically modified.

9. A method for obtaining a cDNA that is a homologous DNA comprising the base sequence set forth in SEQ ID NO: 2 or in SEQ ID NO: 4 in the Sequence Listing, the method comprising:
obtaining the cDNA from a cDNA library with the aid of a polynucleotide as a probe, the cDNA
capable of hybridizing to the polynucleotide used as the probe, wherein the polynucleotide comprises a coding region of the polynucleotide according to any of claims 5 - 8.

10. A cDNA obtained by the method according to claim 9, being a homologous DNA comprising the base sequence set forth in SEQ ID NO: 2 or in SEQ ID NO: 4 in the Sequence Listing.

11. A protein comprising an amino acid sequence that the cDNA according to claim 10 encodes, and being a homologue of the protein according to claim 1 or claim 2.

12. An antibody recognizing the protein according to claim 1, 2, 3, 4 or 11.