JP2000093178A - DD13 gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein which is composed of the protein that contains a specific amino acid sequence and shows metalloendoprotease activity and is the gene product of DD13 gene that expresses after nerve lesion and functions in the process from the nerve lesion to the functional repair. SOLUTION: This protein is a new protein which is composed of the protein specified by the amino acid sequence represented by the formula or the protein that contains the amino acid sequence obtained by deleting, substituting and/or adding one or plurality of amino acids to the amino acid sequence represented by the formula and shows metalloendoprotease activity, consists of the gene product of DD13 gene that expresses and responds after nerve lesion and functions in the process from the nerve lesion to the functional repair and functions as the metalloendoprotease peculiar to nerve systems. This new protein is obtained by screening out the cDNA library prepared by mRNA with the probe consisting of a part of the polynucleotide that codes for the DD13 protein and integrating the resultant DD13 gene into a vector to express in the host cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a gene that controls resistance to nerve damage and a gene product thereof.

[0002]

BACKGROUND OF THE INVENTION Mature central nervous cells have lost the ability to divide and are vulnerable to damage such as ischemia and trauma. Central nerve cells with damaged axons undergo retrograde degeneration, leading to cell death. Peripheral motor nerves, on the other hand, are relatively resistant to injury and survive axonal damage without retrograde degeneration. Further, new axons germinate from the damaged axon ends, project to the target organ again, and achieve functional restoration. Thus, the neurons of the peripheral nervous system and the central nervous system have greatly different resistances to damage, but the molecules responsible for such differences and the regulation of their expression are unknown at present. In order to survive the damaged cells of the central nervous system and aim for functional repair by extending axons, it is first necessary to clarify what causes the resistance of peripheral nerves to damage.

[0003]

An object of the present invention is to provide a gene that controls resistance to peripheral nerve injury. More specifically, it is an object of the present invention to provide a gene capable of expressing and responding after nerve injury and functioning in a process from nerve injury to functional repair. Another object of the present invention is to provide a gene product encoded by the above gene.

The present inventors have made intensive efforts to solve the above-mentioned problems, and as a result, the rat-derived gene specified by SEQ ID NO: 2 in the sequence listing has the above-mentioned characteristics, and is encoded by the gene. It was confirmed that the protein was mainly expressed in neurons, and that it was expressed in large amounts in damaged neurons and involved in the repair of its function. In addition, they have found that the above protein is a metalloprotease specific to the nervous system and is very similar to endothelin converting enzyme. The present invention has been completed based on these findings.

That is, the present invention provides a protein specified by the amino acid sequence of SEQ ID NO: 1 in the Sequence Listing. Also provided is a protein comprising the amino acid sequence of SEQ ID NO: 1 in which 1 or several amino acids are deleted, substituted, and / or added, and having a metalloendoprotease activity. .

In another aspect, the present invention provides a protein comprising a partial sequence of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing,
A protein having metalloendoprotease activity; and
Provided is a protein comprising a partial sequence of an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added to the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, and having a metalloendoprotease activity. . These proteins are provided, for example, in the form of a soluble protein having a function as an active domain.

[0007] In another aspect, the invention comprises an amino acid sequence containing one of the above amino acid sequences as a partial sequence,
And a protein having metalloendoprotease activity. This protein is provided, for example, in the form of an extracellular secretory protein containing the above-mentioned protein as an active domain and a signal peptide. The term “protein of the present invention” used herein is used as a concept including any of the above-described proteins unless otherwise specified.

[0008] From another viewpoint, an antibody that recognizes the protein of the present invention is also provided. The antibodies include polyclonal antibodies, monoclonal antibodies, and active fragments thereof. Further, a polynucleotide encoding the protein of the present invention is provided. According to a preferred embodiment, 96 of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing
A DNA comprising a base sequence from the Ath position to the 2420th G position is provided. According to a further preferred embodiment, a continuous one of the nucleotide sequences described in SEQ ID NO: 2 in the sequence listing.
A DNA consisting of two or more bases is provided. This DNA
May be either double-stranded or single-stranded, and in the case of single-stranded, may be either sense strand or antisense strand. An RNA that hybridizes to the above DNA;
A polynucleotide obtained by chemically modifying the above polynucleotide is also provided by the present invention.

Further, according to the present invention, the above protein is isolated from a recombinant vector containing the above-mentioned polynucleotide, a transformant such as a microbial cell or a mammalian cell containing the vector, and a culture in which the transformant is cultured. -There is provided a method for producing the above protein, comprising a step of purifying.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The DD13 protein of the present invention is a rat-derived protein, and is characterized by comprising the amino acid sequence of SEQ ID NO: 1 in the sequence listing. In the amino acid sequence of SEQ ID NO: 1 in the sequence listing, one or several,
A protein comprising an amino acid sequence in which preferably 1 to 20, more preferably 1 to 10 amino acids have been deleted, substituted and / or added, and having a metalloendoprotease activity is also included in the scope of the present invention. (This protein is sometimes referred to as “DD13 mutant”.)

The DD13 mutant is obtained, for example, by subjecting E. coli or the like having a DNA encoding the DD13 protein to mutation treatment using an agent such as N-nitro-N'-nitro-N-nitrosoguanidine, and transforming the DD13 mutant from the cells. After the gene encoding the body is recovered, it can be produced by performing a normal gene expression operation. Alternatively, the gene may be directly treated with a drug such as sodium sulfite, or site-directed mutagenesis (Kramer, W. et al., Methods in Enzymol
ogy, 154, 350, 1987) and recombinant PCR (PCR
Technology, Stockton press, 1989), directly introducing nucleotide deletions, substitutions or additions.

The method for obtaining the gene of the present invention encoding the DD13 protein (hereinafter sometimes referred to as “DD13 gene”) is not particularly limited. For example, a cDNA that hybridizes to the above gene from a cDNA library may be used. Is specifically described. However, the method for obtaining the gene of the present invention is not limited to the following method.

1) A DNA consisting of the coding region of the nucleotide sequence of SEQ ID NO: 1 or 3 was prepared using Takara MEGA
Label using a 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- ³² P] ATP (370 MBq / ml) (Amersham) 5 μl T4 polynucleotide kinase 1 μl Total 10 μl

3) TE (50 mM Tris-Cl p
H8.0, 1 mM EDTA)
adexG-25 (registered trademark, manufactured by Pharmacia) is packed in a 1.5 ml polyprep column (manufactured by Bio-Rad) so that the bed volume becomes 1 ml, and the DNA reaction solution that has been heat-treated in 2) is loaded on the column. 4) After that, 200 μl T ₅₀ E was passed through the column four times,
A labeled DNA probe is obtained from the fraction eluted with 200 μl of the second time.

5) From the labeled DNA probe fraction obtained above, 150 μl of the cDNA in a plaque immobilized on a nitrocellulose membrane [immobilization of a cDNA library on a nitrocellulose membrane was performed by using Molecular
Cloning 2nd Edition (Cold
Spring Harbor LaboratoryP
Res., 1989) Chapter 9, pages 9.38 to 9.4
It can be performed according to the method described on page 1. ] And the following conditions. Prehybridization 6 × SSC 5 × Denhardt's 0.05% sodium pyrophosphate 100 μg / ml denatured herrin
g 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 herrin
g 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 autoradiogram. 8) From the obtained autoradiogram, determine the position of the positive plaque, and collect the corresponding plaque on agar in the 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) The above steps 5) to 9) are repeated three times to make a single positive plaque. The plaque was collected and 10
Suspend in 0 μl SM solution to stabilize phage.
The gene isolated from the plaque is cDNA that hybridizes to DD13 DNA.

CD hybridizing to DD13 gene
Large scale preparation of NA 1) 50 μl of plaque phage suspended in SM solution and 20 μl of Y1090r-E. Coli were mixed at 37 ° C.
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 (Tris-Cl pH 8.0 10m
M, 1 mM EDTA) to obtain a DNA solution.

The degeneracy of the genetic code allows substitution of at least a part of the base sequence of the polynucleotide with another type of base without changing the amino acid sequence of the polypeptide produced from the polynucleotide. Is well known to those skilled in the art. Therefore, the polynucleotide of the present invention includes all polynucleotides encoding the DD13 protein or the DD13 mutant. As a preferred example of the gene of the present invention, the gene encoding the DD13 protein derived from rat is shown in SEQ ID NO: 2 in the sequence listing. In some cases, a gene encoding a DD13 mutant can be obtained by the above method. The amino acid sequence of the DD13 mutant can be determined from the nucleotide sequence of the gene encoding the mutant. For example, it can be performed using a commercially available program (for example, MacVector (registered trademark, manufactured by Eastman Chemical Company)).

The scope of the present invention includes antisense polynucleotides comprising the nucleotide sequence of the antisense strand of the polynucleotide encoding the DD13 protein and derivatives thereof. The antisense polynucleotide is provided as one embodiment of the above-mentioned polynucleotide. is there. The antisense polynucleotide is capable of hybridizing to a polynucleotide encoding the DD13 protein, and if the hybridizing polynucleotide is a polynucleotide of a coding region, the polynucleotide of the polypeptide encoded by the polynucleotide may be used. It is possible to inhibit biosynthesis.

The antisense polynucleotide for inhibiting the biosynthesis of the polypeptide preferably comprises 12 or more bases. On the other hand, sequences that are longer than necessary are not preferred in order to incorporate the full-length antisense polynucleotide into cells. When incorporating the antisense polynucleotide into cells and inhibiting the biosynthesis of the DD13 protein, a base having 12 to 30 bases, preferably 15 to 25 bases, more preferably 18 to 22 bases It is preferable to use an antisense polynucleotide consisting of

The antisense polynucleotides or derivatives thereof of the present invention include all those in which a plurality of nucleotides consisting of a base, a phosphate, and a sugar are linked, regardless of whether they are naturally occurring or not. Typical are natural antisense DNA and antisense RNA. Examples of the non-naturally-occurring polynucleotide include a polynucleotide of a methylphosphonate type and a phosphorothioate type. The antisense polynucleotide of the present invention can be prepared using the antisense technology available to those skilled in the art using the DNA or mR of interest.
Various antisense polynucleotide derivatives having excellent binding ability to NA, tissue selectivity, cell permeability, nuclease resistance, intracellular stability and the like can be obtained.

In general, from the viewpoint of ease of hybridization, it is preferable 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. . Therefore, the antisense polynucleotide of the present invention and its derivative can form a stem loop as needed, and such an embodiment is a preferable example of the antisense polynucleotide of the present invention. 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, wherein the translation initiation codon of the gene or mRNA encoding the DD13 protein,
Those containing a sequence complementary to the ribosome binding site, capping site, and / or splice site are preferred embodiments from the viewpoint of the expression suppressing effect.

Among the generally known polynucleotide derivatives, derivatives having at least one of enhanced nuclease resistance, tissue selectivity, cell permeability and binding strength, preferably have a phosphorothioate bond as a backbone. Derivatives having a structure can be given. The polynucleotides and derivatives thereof of the present invention include derivatives having these functions or structures.

Of the antisense polynucleotides of the present invention, natural antisense polynucleotides may be synthesized using a chemical synthesizer or produced by PCR using a DNA encoding the DD13 protein as a template. Can be. A polynucleotide derivative such as a methylphosphonate type or a phosphorothioate type can usually be produced by chemical synthesis. In this case, the operation can be performed according to the instructions attached to the chemical synthesizer, and the obtained synthetic product can be purified by an HPLC method using reverse phase chromatography or the like.

The polynucleotide encoding the DD13 protein of the present invention, its antisense polynucleotide or a part thereof (polynucleotide having a continuous base sequence of 12 or more bases) is c-type.
It can be used as a probe for screening a DNA encoding the DD13 protein from a DNA library or the like. For such a purpose, 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.

[0029] As a cDNA library used in screening using the probe, one prepared from mRNA can be preferably used. These cds
A group of cDNAs selected by random sampling from the NA library can be used as a sample for the search. Also, commercially available products can be used. For example, 12 consecutive nucleotides in the base sequence described in SEQ ID NO: 2 in the sequence listing can be used.
DNA consisting of a base sequence of bases or more or a polynucleotide hybridizing to the DNA (antisense polynucleotide) is a DNA encoding the DD13 protein.
Can be used as a probe for screening from a cDNA library or the like.

Further, Northern blot hybridization is performed on mRNA derived from each tissue using a polynucleotide encoding the DD13 protein of the present invention, an antisense polynucleotide thereof, or a polynucleotide which is a part thereof as a probe. It is possible to find tissues in which mRNA derived from DD13 gene is expressed.

The cDNA that hybridizes with the DD13 gene obtained above is inserted into an appropriate vector (for example, pME18).
After insertion into a host (for example, Escherichia coli), a transformant can be prepared. The type of the vector and the type of the host are not particularly limited, but an appropriate expression vector can be selected and used according to the type of the host. As a host, bacteria such as E. coli,
Either yeast or animal cells can be used. A method for obtaining a transformant by introducing a recombinant vector into an appropriate host such as Escherichia coli is not particularly limited, and any method available to those skilled in the art can be applied.

The DD13 protein can be produced by culturing the transformant into which the DD13 gene of the present invention has been introduced and amplifying the gene DNA or expressing the protein. There are various books and reports on the production and culture of transformants, and various means have been developed and widely used in the art. Therefore, those skilled in the art can easily produce DD13 protein based on the nucleotide sequence described in the present specification. As a technique for introducing a gene into cells, a calcium chloride method, a lipofection method, a protoplast method, an electroporation method, or the like can be used.

The target protein can be separated and purified from the culture by appropriately combining means available to those skilled in the art. For example, the DD13 protein of the present invention can be efficiently recovered and purified by performing operations such as concentration, solubilization, dialysis, and various types of chromatography, if necessary. More specifically, immunoprecipitation, salting out, ultrafiltration, isoelectric precipitation, gel filtration, electrophoresis, ion exchange chromatography, hydrophobic chromatography, antibody chromatography, etc. Any of affinity chromatography, chromatofocusing method, adsorption chromatography method, reverse phase chromatography method, and the like may be appropriately selected and performed. By using the gene encoding the DD13 mutant, the DD13 mutant can be similarly produced.

The DD13 protein or DD13 mutant can also be produced as a fusion peptide with another polypeptide. Such fusion peptides are also included in the scope of the present invention. The type of polypeptide to be fused is not particularly limited, and examples include a signal peptide that promotes extracellular secretion. Production of such a fusion protein can also be performed using a transformant. When a DD13 protein or a DD13 mutant is produced using the fusion protein, the fusion protein may be treated with a chemical substance such as bromocyan or an enzyme such as a prosthesis, and the cut out target product may be separated and purified.

Further, it is possible to produce a fusion protein of a partial polypeptide (so-called active domain) responsible for the metalloendoprotease activity in the DD13 protein or the DD13 mutant and another polypeptide. Examples of such active domains include DD13 proteins and polypeptides of DD13 mutants excluding the hydrophobic portion. For example, in the DD13 protein derived from rat described in SEQ ID NO: 1 in the sequence listing, the transmembrane domain is presumed to be located at amino acids 62 to 85 of the amino acid sequence (see FIG. 3). By removing the N-terminal polypeptide containing, a soluble polypeptide containing the active domain can be produced. A fusion protein of the active domain solubilized in this way and another polypeptide (for example, a signal peptide) is also included in the scope of the present invention.

The DD13 protein or DD13 of the present invention
Using variants or their partial polypeptide chains, D
An antibody recognizing the D13 protein or the modified DD13 (hereinafter, sometimes referred to as “DD13 antibody”) can be produced. The antibodies of the present invention are used to
By immunizing with D13 protein or DD13 variant, it can be produced according to a method commonly used in the art. Whether the antibody recognizes the DD13 protein or the modified DD13 of the present invention can be confirmed by Western blotting, ELISA, immunostaining (for example, measurement by FACS), or the like. As the immunogen, in addition to the DD13 protein or the DD13 mutant, one obtained by binding a part thereof to another carrier protein such as bovine serum albumin may be used. The DD13 protein or a part of the DD13 mutant preferably has 8 amino acid residues or more, and such a polypeptide may be synthesized using, for example, a peptide synthesizer.

A monoclonal antibody produced from a hybridoma produced using lymphocytes of an immunized animal may be used as the antibody of the present invention. Methods for producing monoclonal antibodies are well known in the art and are widely used ("Antibodies A La").
"Boratory Manual" (Cold Spr
ing Harbor Laboratory Pre
ss, 1988) Chapter 6). Further, as the antibody of the present invention, an active fragment of the above antibody can be used. In the present specification, the active fragment means a fragment of an antibody having an antigen-antibody reaction activity, and specifically, F (ab ') 2, Fab',
Fab, Fv and the like can be mentioned. 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 (a
b ′) 2 is reduced with a reagent such as 2-mercaptoethanol and alkylated with monoiodoacetic acid to give 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 above-described antibodies and active fragments are all included in the scope of the present invention.

The protein of the present invention can be detected according to a method using an antibody or a method using an enzymatic reaction.
Methods for detecting DD13 protein using an antibody include:
Specifically, using (I) labeled DD13 antibody,
(II) DD13 antibody and a method of detecting DD13 protein using a labeled secondary antibody of the antibody. As the label, for example, a radioisotope (RI), an enzyme, avidin or biotin, or a fluorescent substance (FITC, rhodamine, or the like) is used. As a method using an enzyme reaction, for example, an ELISA method,
Examples include a method for identifying an immunoreactive molecule using an immunoagglutination method, a Western blot method, and flow cytometry, or a method similar thereto. The protein of the present invention, preferably DD13 protein, is classified as a metalloendoprotease. For example, it can function as a metalloendoprotease specific to the nervous system.

[0039]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited to the following examples. Animal surgery 70 Wistar rats weighing approximately 100 g were pentobarbital
(45 mg / kg), the right hypoglossal nerve was cut with scissors in the supine position, and then the hypoglossal nucleus was incised from the operated side and the normal side. Collected 70 sublingual nuclei (operated and normal),
Frozen in liquid nitrogen. Total RNA (about 70 mg) was collected from the operated sublingual nucleus or normal sublingual nucleus by the AGPC method.

The differential display PCR (DD-
PCR) Total RNA (0.2 μg each) was transferred to Superscript Reverse Transcriptase (GI
BCO / BRL) and cDNA using nucleotide oligo dT18
Inverted. Subsequently, 1/10 amount of each cDNA pool was added to [α- ³⁵S] dA
TP (New England Nuclear-Dupont, Nati
And MA) in the presence of any of the 87 primers
Selected single primer (5'-ACTGGCCGAGGG-
Amplified by PCR using 3 '). Cycle parameters
Are as follows: denaturation at 94 ° C. for 5 minutes;
Denaturation by 0 seconds, annealing at 42 ° C. for 1 minute, and 72
Elongation at 1.5 ° C for 1.5 minutes for a total of 40 cycles;
Continue with additional extension at 72 ° C for 5 minutes. Radioactivity label
Electrophoresed PCR products on a 5% sequencing gel
And visualized by autoradiography (Fig.
1). Increased expression on the sample from the nucleus
Recover bands from denatured polyacrylamide gel
And recycle by 40-cycle PCR using the same primers.
Amplified. TA cloning kit for reamplified cDNA products
(Invitrogen, San Diego, CA) using PCRTMI
Cloned into the I vector.

Cloning of DD13 A partial 200 bp cDNA encoding the DD13 protein was obtained by differential display PCR. DD13 full length cDNA
5 ′ and 3 ′ RACE reactions were performed to isolate

5 ′ RACE 4 μg of total RNA from the rat sublingual nucleus on the operated side was dissolved in water and denatured at 70 ° C. for 5 minutes. The first linear cDNA is DD-PCR
The fragment was synthesized by treating with Superscript ^TM reverse transcriptase (GIBCO / BRL) at 42 ° C. for 1 hour using fragment-specific primers. Anchor oligomer (5'-PO ₄ -GTAGGAATTC
GGGTTGTAGGGAGGTCGACATTGCC-NH ₂ -3 ′) was added to the 3 ′ end by T4 RNA ligase. After incubating at 42 ° C. for 1 hour, the temperature was increased to 51 ° C. and the RT reaction continued for another 30 minutes. Then the RNA is treated with 4U RNaseH (TOYOBO) to 37
C. for 30 minutes. The RT reaction was purified by phenol: chloroform extraction. First round PCR is LA taq
DD-PCR fragment-specific 3 'primer and anchor B primer (GGCA) using polymerase (TAKARA)
ATGTCGACCTCCCTACAAC). PCR conditions are as follows: denaturation at 98 ° C. for 20 seconds, annealing and extension at 68 ° C. for 4 minutes for a total of 30 cycles. Nested PCR reaction with another specific primer and primer C (CTCCCTACAACCC
GAATTCCTAC). The 5'RACE product was subcloned into the pGEM-T vector (Promuga) and sequenced on an Applied Biosystems model 373A sequencer. Both strands of the cDNA were covered at least twice. Based on the sequence of the obtained 5'RACE product, three more 5'RACEs were performed until the full-length cDNA encoding the DD13 protein was isolated.
Was done.

3 ′ RACE 4 μg of total RNA from the rat sublingual nucleus on the operated side was used.
The first chain cDNA is GAGTCGACTCGAGAATTTC- (T) ₇ Superscript ^TM reverse transcriptase using oligo primers (G
IBCO / BRL). The first 3 'RACE reaction is DD-PCR
Performed with a specific 5 'primer and a GAGTCGACTCGAGAATTC primer. One-sided nested PCR reactions were performed using another specific primer and the GAGTCGACTCGAGAATTC primer.

The obtained 5 'and 3' RACE products are single mRN
The following experiment was performed in order to confirm whether or not it was derived from A. RT-PCR The amplified PCR product encoded the entire sequence of the obtained rat DD13 ORF. Mouse Genome Library Screening A mouse genome library (λFIXII library, Stratagene, La Jolla, CA) was screened with the subcloned PCR-derived DNA fragment to obtain the DD13 mouse genome. The probe was labeled [α] using a multiprime DNA labeling system (Amersham, UK).
- was labeled with ³² P] dCTP (Amushamu, UK). About 1
× 10 ⁶ plaques were spread to a density of 1 × 10 ⁴ pfu / 150 mm dish and screened under the following hybridization conditions: hybridization was 6 × SS
C, in 5 × Denhardt's solution and 0.5% SDS, the concentration of 1 × 10 ⁶ cpm / ml, and treated at 65 ° C., washing 2 × SSC / 0.5% SDS and 0.1 ×
Performed at 65 ° C. in SSC / 0.5% SDS. Five positive plaques were obtained, which were purified and partially sequenced. These phage clones contained a start codon and a stop codon in the rat DD13 sequence.

Putative amino acid sequence of rat DD13 The putative transmembrane domain was located at positions 62-85 of the rat DD13 amino acid sequence. A conserved zinc binding motif was found at positions 612-616 (FIG. 2).

Northern hybridization Total RNA was extracted from the normal sublingual nucleus of rats and the operated sublingual nucleus by the AGPC method. For Northern analysis, 20 μg of total RNA was separated by formaldehyde / 0.8% agarose gel electrophoresis and transferred to a Hybond N + membrane (Amersham Corporation). T7 polymerase and ^alpha-32 P UTP
(New England Nuclear-Dupont, Natick, MA) using DD13 cDNA (396-477 aa and DD-PCR).
2 types of ³² P by in vitro transcription
-A labeled RNA probe was prepared. Hybridization is performed using hybridization buffer (5 × 10 ⁶ c
pm / ml labeled probe, 50% formamide, 10% dextran sulfate, 1.5 × SSPE, 1% SDS, 0.5% blot, 0.2 mg / ml yeast RNA, 0.5 mg / ml salmon sperm DNA), 55
C. was performed.

The above-mentioned membrane was prepared by adding 2 × SSC / 0.1% SDS, 0.5 × S
Wash with SC / 0.1% SDS, 0.1 × SSC / 0.1% SDS, rinse with PBS, incubate in RNase buffer containing RNaseA (10 μg / ml), rinse with PBS and autoradiography at −80 ° C. for 1 day To FIG. 4 shows the results. A shows the results of hybridization with a probe derived from the differential display fragment. A single band was observed at 7.5 kb. B shows the results of hybridization with the probe corresponding to DD13 ORF. The major band was 3.5 kb in size, but a faint band 7.5 kb in size was also observed.

Expression of DD13 protein in HEK293T cells cDNA encoding rat DD13 protein was expressed in expression vector pME1
Insertions in 8S were transfected into HEK293T cells using FuGENE ^™ 6 (Boehringer Mannheim). 50 transfected with pME18S-DD13 and pME18S
After time, cells were harvested, washed with phosphate buffered saline, 20 mM Tris-HCl, pH 7.5, 5 mM MgCl ₂ , 0.1 mM phenylmethylsulfonyl fluoride, 20 μM pepstatin, 2 mM
Homogenized in 0 μM leupeptin. The homogenate was centrifuged at 15,000 rpm for 45 minutes. 0.5% of the obtained membrane fraction
(w / v) It was dissolved in a homogenizing buffer containing Triton X-100. After stirring this sample for 30 minutes, 15,000
Centrifuged at rpm for 60 minutes. The supernatant was used for subsequent analysis.

DD13 Polyclonal Antibodies Antibodies to DD13 were prepared by immunizing rabbits with a synthetic peptide, MTAHYDEFQEVKYESRC, corresponding to 7-23 aa of the rat DD13 protein. Rabbits were immunized four times with KLH-coupled peptide with complete adjuvant at two-week intervals, after which the antisera was affinity purified (TANA Laboratories, LC).

Western blotting The collected membrane proteins were used for immunoblotting. These were separated on a 7% SDS / PAGE gel and transferred to a PVDF membrane (Bio-Rad). Blot
Block with 5% (w / v) dry milk in PBS for 30 minutes, D
The plate was incubated with the D13-671 rabbit polyclonal antibody, washed with PBS containing 0.1% Tween 20, and incubated with alkaline phosphatase-conjugated anti-rabbit IgG diluted 1: 5000. Incubation with the primary and secondary antibodies was both performed for 1 hour at room temperature. Immunoreactive bands were visualized by alkaline phosphatase enzyme reaction. Immunoblot analysis using 671 Ab revealed a strong band in the membrane fraction prepared from DD13 transfected cells, but not in cells transfected with the vector alone. This band was located at about 100 kDa (FIG. 5).

In Situ Hybridization Animals were dissected from the neck and their brains were immediately removed and frozen in finely ground dry ice. 20 μm on cryostat
Prepared on a glass slide coated with 3-aminopropyltriethoxysilane,
Stored at -80 ° C until use. Immediately before use, sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 20 minutes, washed with phosphate buffer, and then washed with 50 mM Tris-HCl.
And treated with Protease-K (10 μg / ml) in 5 mM EDTA for 10 minutes and then re-fixed. After washing the sections with distilled water, acetic anhydride (0.2 M) in 0.1 M triethanolamine was used.
5%), rinsed with phosphate buffer, treated stepwise with concentrated ethanol and dehydrated (70%, 95% and
100%). Then, it was defatted with chloroform, washed with ethanol and air-dried.

[0052] and SP6 or T7RNA polymerase [α- ³⁵ S] UTP
(New England Nuclair-Dupont)
An RNA probe labeled with ³⁵ S was prepared by an in vitro transcription method. Hybridization buffer (50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, 5 mM EDT
A, 10 mM phosphate buffer, 10% dextran sulfate, 1 × Denhardt's solution, 0.2% sarkosyl, 500 μg / ml yeast transfer RNA, and 200 μg / ml salmon sperm DN
A) The probe labeled in (5 × 10 ⁶ cpm / ml) was
After denaturation at ℃, they were mounted on sections.

Hybridization was carried out overnight in a 55 ° C. constant humidity bath. The hybridized section was subjected to 5 x SSC at 55 ° C.
-1% Rinse lightly with 2-mercaptoethanol, 50% at 60 ° C
Washed with deionized formamide, 2 × SSC and 10% 2-mercaptoethanol (high stringency buffer) for 30 minutes. RNase buffer (0.5M NaCl, 10mT Tris-H
The sections were rinsed with Cl and 1 mM EDTA, reacted with RNase-A (1 μg / ml) in RNase buffer at 37 ° C. for 30 minutes, and rinsed with RNase buffer. The sections were then incubated in the above buffer, rinsed in 2 × SSC and 0.1 × SSC at room temperature for 10 minutes each, dehydrated stepwise using concentrated ethanol, and air-dried.

The sections were exposed to an X-ray film for 3-6 days and then immersed in IIfold K-5 photoemulsion (IIfold) diluted 6: 4 with water. The sections were exposed to the emulsion for 1-2 weeks in the dark at 4 ° C., developed with Kodak D19 developer, the sections were counterstained with thionin, dehydrated with ethanol and xylene, placed on a cover glass, and observed under a microscope. did. FIG. 6 shows that levels of DD13 mRNA are elevated after sublingual nerve axotomy and ischemia. The DD13 protein is strongly expressed in the nervous system during the fetal period, and a positive signal of DD13 was also observed in intestinal ganglion neurons (FIG. 7). Thus, the DD13 gene is thought to respond to various brain injuries.

RNase protection assay RPAII ribonuclease protection assay kit
(Ambion) was used for RNase protection assay. The cRNA probe was prepared by cloning DD13 cDNA into the pBluescript KS vector, linearizing it, and in vitro transcription with T7 RNA polymerase and ³² P-UTP. Total RNA (10 μg) from various tissues was hybridized overnight at 45 ° C. to the labeled probe. Reactions were digested with RNaseA / T1, electrophoresed on a 4% sequencing gel and visualized by autoradiography for 1 day. As a result, it was shown that the DD13 protein was specifically expressed in the nervous system (FIG. 8).

[0056]

[Sequence List] Sequence Listing <110> Sumitomo Electric Industries, Co., Ltd. <120> DD13 GENE <130> 98 128M <160> 2 <210> 1 <211> 775 <212> PRT <213> Rat <400> 1 Met Glu Ala Pro Tyr Ser Met Thr Ala His Tyr Asp Glu Phe Gln Glu 5 10 15 Val Lys Tyr Glu Ser Arg Cys Gly Thr Gly Gly Ala Arg Gly Thr Ser 20 25 30 Leu Pro Pro Gly Phe Pro Arg Ser Ser Gly Arg Ser Ala Ser Gly Ala 35 40 45 Arg Ser Gly Leu Pro Arg Trp Asn Arg Arg Glu Val Cys Leu Leu Ser 50 55 60 Gly Leu Val Phe Ala Ala Gly Leu Cys Ala Ile Leu Ala Ala Met Leu 65 70 75 80 Ala Leu Lys Tyr Leu Gly Pro Gly Ala Ala Gly Thr Gly Gly Ala Cys 85 90 95 Pro Glu Gly Cys Pro Glu Arg Lys Ala Phe Ala Arg Ala Ala Arg Phe 100 105 110 Leu Ser Ala Asn Leu Asp Ala Ser Ile Asp Pro Cys Gln Asp Phe Tyr 115 120 125 Ser Phe Ala Cys Gly Gly Trp Leu Arg Arg His Ala Ile Pro Asp Asp 130 135 140 Lys Leu Thr Tyr Gly Thr Ile Ala Ala Ile Gly Glu Gln Asn Glu Glu 145 150 155 160 Arg Leu Arg Arg Leu Leu Ala Arg Pro Thr Gly Gly Pro Gly Gly Ala 165 170 175 Ala Gln Arg Ly s Val Arg Ala Phe Phe Arg Ser Cys Leu Asp Met Arg 180 185 190 Glu Ile Glu Arg Leu Gly Pro Arg Pro Met Leu Glu Val Ile Glu Asp 195 200 205 Cys Gly Gly Trp Asp Leu Gly Gly Ala Ala Asp Arg Pro Gly Ala Ala 210 215 220 Arg Trp Asp Leu Asn Arg Leu Leu Tyr Lys Ala Gln Gly Val Tyr Ser 225 230 235 240 Ala Ala Ala Leu Phe Ser Leu Thr Val Ser Leu Asp Asp Arg Asn Ser 245 250 255 Ser Arg Tyr Val Ile Arg Ile Asp Gln Asp Gly Leu Thr Leu Pro Glu 260 265 270 270 Arg Thr Leu Tyr Leu Ala Gln Asp Glu Gly Ser Glu Lys Val Leu Ala 275 280 285 Ala Tyr Lys Val Phe Met Glu Arg Leu Leu Arg Leu Leu Gly Ala Asp 290 295 300 Ala Val Glu Gln Lys Ala Gln Glu Ile Leu Gln Leu Glu Gln Arg Leu 305 310 315 320 Ala Asn Ile Ser Val Ser Glu Tyr Asp Asp Leu Arg Arg Asp Val Ser 325 330 335 Ser Val Tyr Asn Lys Val Thr Leu Gly Gln Leu Gln Lys Ile Thr Pro 340 345 350 His Leu Gln Trp Lys Trp Leu Leu Asp Gln Ile Phe Gln Glu Asp Phe 355 360 365 Ser Glu Glu Glu Glu Val Val Leu Leu Ala Thr Asp Tyr Met Gln Gln Gln 370 375 380 Val Ser Gln Leu Il e Arg Ser Thr Pro Arg Arg Ile Leu His Asn Tyr 385 390 395 400 400 Leu Val Trp Arg Val Leu Val Val Leu Ser Glu His Leu Ser Pro Pro 405 410 415 Phe Arg Glu Ala Leu His Glu Leu Ala Lys Glu Met Glu Gly Asn Asp 420 425 430 Lys Pro Gln Glu Leu Ala Arg Val Cys Leu Gly Gln Ala Asn Arg His 435 440 445 Phe Gly Met Ala Leu Gly Ala Leu Phe Val His Glu His Phe Ser Ala 450 455 460 Ala Ser Lys Ala Lys Val Gln Gln Leu Val Glu Asp Ile Lys Tyr Ile 465 470 475 480 480 Leu Gly Gln Arg Leu Glu Glu Leu Asp Trp Met Asp Ala Gln Thr Lys 485 490 495 Ala Ala Ala ARG Ala Lys Leu Gln Tyr Met Met Val Met Val Gly Tyr 500 505 510 510 Pro Asp Phe Leu Leu Lys Pro Glu Ala Val Asp Lys Glu Tyr Glu Phe 515 520 525 Glu Val His Glu Lys Thr Tyr Leu Lys Asn Ile Leu Asn Ser Ile Arg 530 535 540 Phe Ser Ile Gln Leu Ser Val Lys Lys Ile Arg Gln Glu Val Asp Lys 545 550 555 560 Ser Thr Trp Leu Leu Pro Pro Gln Ala Leu Asn Ala Tyr Tyr Leu Pro 565 570 575 Asn Lys Asn Gln Met Val Phe Pro Ala Gly Ile Leu Gln Pro Thr Leu 580 585 590 Tyr Asp Pro Asp Ph e Pro Gln Ser Leu Asn Tyr Gly Gly Ile Gly Thr 595 600 605 Ile Ile Gly His Glu Leu Thr His Gly Tyr Asp Asp Trp Gly Gly Gln 610 615 620 Tyr Asp Arg Ser Gly Asn Leu Leu His Trp Trp Thr Glu Ala Ser Tyr 625 630 635 640 Ser Arg Phe Leu His Lys Ala Glu Cys Ile Val Arg Leu Tyr Asp Asn 645 650 655 Phe Thr Val Tyr Asn Gln Arg Val Asn Gly Lys His Thr Leu Gly Glu 660 665 670 Asn Ile Ala Asp Met Gly Gly Leu Lys Leu Ala Tyr Tyr Ala Tyr Gln 675 680 685 Lys Trp Val Arg Glu His Gly Pro Glu His Pro Leu His Arg Leu Lys 690 695 700 Tyr Thr His Asn Gln Leu Phe Phe Ile Ala Phe Ala Gln Asn Trp Cys 705 710 715 715 720 Ile Lys Arg ARG Ser Gln Ser Ile Tyr Leu Gln Val Leu Thr Asp Lys 725 730 735 His Ala Pro Glu His Tyr Arg Val Leu Gly Ser Val Ser Gln Phe Glu 740 745 750 Glu Phe Gly Arg Ala Phe His Cys Pro Lys Asp Ser Pro Met Asn Pro 755 760 765 Val His Lys Cys Ser Val Trp 770 775 <210> 2 <211> 7507 <212> DNA <213> Rat <400> 2 gaccgagggc catgtgggcg ccgaccagaa agcccgcaga cagaagcaac ccataactga 60 ccaaacaagg ggtctgcagc caaagcctcg gcaca atg gag gcc ccg tat tcc 113 atg aca gcg cac tat gat gag ttc cag gaa gtc aag tac gag agc cgg 161 tgt ggc acc ggg ggc gcc cga ggg acc tcc cta cct cca gg c gg ag c cc g c gg ag c gg ag c cc c gc cc gc cc g agc ggg gcc cgg tcg ggg cta ccg cgc 257 tgg aac cga cgg gag gtg tgc ctg ttg tcc ggg ctg gtg ttc gcc gcc 305 ggc ctc tgt gcc atc ttg gca gcc atg ctg gc g ct cg c c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g c g ggc gga gcc tgt ccc gag ggc tgc ccc gag 401 cgc aag gcc ttc gcg cgc gcc gcc cgc ttc ctg tct gcc aac ctg gac 449 gcc agc atc gac ccg tgc cag gac ttc tac tc g cg cc gc cc gc cc gc cc gc cg cc gc cc gc cg cc gc cg cc gc cc gc gcc atc ccc gac gac aag ctc acc tat ggc acg 545 atc gcg gcc atc gga gag cag aac gag gag cgc ctg cgg cgc ctg ctg 593 gcg aga ccc acg gga ggc ccg ggc ggc gc gc cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg cg gc tcg tgt ctc gac atg cgt gag atc gag agg ctc ggg 689 ccg cgg ccc atg ctg gag gtc atc gag gac tgc ggc ggc tgg gac cta 737 ggc ggc gcg gcc gat cgc ccg ggg gc g gc cg cg cg gc gc gc gc gc cg cg ttg tac aag gca cag ggt gtg tac agc gcg gcc gcg ctc ttc tcg 833 ctt aca gtc agc ctg gac gac agg aac tcc tcg cgc tac gtc atc cgt 881 att gac cag gac ggg ctc acc ct c gac ctc acc ct c gac ctc acc cc c gc caa gat gag ggg agt gag aag gtc ctg gcc gca tac aag gtg ttc atg977 gg 1073 gaa tat gat gat ctc cgt cga gat gtc agc tcc gtg tac aac aaa gtg 1121 act ctg ggg cag ctg cag aag atc acc ccc cat ttg cag tgg aaa tgg 1169 ctg ctg gac cag atc ttc cag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gag gtg 1217 gtg ctg ctg gcc aca gac tac atg cag cag gtg tcc cag ctc atc cga 1265 tcc aca ccc cga agg atc ctt cac aac tac cta gtg tgg cgt gtg gtg 1313 gtg gtt ctg agt gag cac gc gc cca cg ctg cat 1361 gag ctg gcc aaa gag atg gag ggc aat gac aaa ccc cag gag ttg gcc 1409 cga gtc tgc ctg ggc cag gcc aac cgc cac ttc ggc atg gct ctt ggt 1457 gcc ctc ttt gta cacga cc agt aaa gcc aag gta 1505 cag cag ctg gtg gaa gat atc aag tac atc tta ggc cag cgc ctg gag 1553 gag ctg gac tgg atg gac gcc cag acc aag gca gct gct cgg gct aag 1601 ctc cag tac atg atg tat cca gac ttc ctg ctg aaa 1649 cct gag gct gta gac aaa gag tat gag ttt gag gtc cac gag aag acc 1697 tac ctc aag aac att ttg aac agc atc cga ttc agc atc cag ttg tct 1745 ggtg ag gc ag gc cag gac aaa tcc acg tgg ctg ctc cct 1793 ccg cag gcg ctc aat gcc tac tat ctg ccc aac aag aac caa atg gta 1841 ttc cca gct ggc atc ctg cag ccc acc ctg tat gac cca gac ttc gc ttc gc ttc gc ttc gc ttc ccg gtt atc ggt acc atc att ggg cat gaa ctg 1937 acc cac ggc tat gat gac tgg gga gga cag tat gac cgc tca gga aac 1985 ctg ctg cac tgg tgg act gag gcc tcc tac agc cgc ttt ctg cac aag 2033 gcc gtt ctc tat gac aac ttc acc gtc tac aac cag 2081 cgg gtg aat ggg aaa cac aca ctt ggc gag aac atc gca gac atg gga 2129 ggc ctc aag ctg gcc tac tat gcc tat cag aag tgg gtc cgg gag cat 177 a gaa cac ccg ctg cac cgg ctc aag tac acc cac aac cag ctt 2225 ttc ttc att gcc ttt gcc cag aac tgg tgc atc aag cga cgg tca cag 2273 tcc atc tac ctg cag gtg ctg aca gac cac gac cac gac cac gac cac gac cac cac gac cac gac cac gac cac gac cac gac cac cac cgg gtc ctg ggt agc gtg tcc cag ttt gag gaa ttc ggc cgg gcc ttc 2369 cac tgt ccc aag gac tct ccc atg aac cct gtc cat aaa tgc tct gtg 2417 tgg tga gcctgtctac ctgcacgtcc ccactgccct gcacggatca cctctgccag 2473 ctgctggggc aacgtcagtc ccctatgtcc ctgctccaga tcgccttcct ccgtccccca 2533 tagcacgaag gaactttagg gtgacacagg gacttgctgg caatggaaag ccaggtttgt 2593 tttttcagtc ttccagctgg gcctcaacgt cagattctcc tttgggaagg tgtctggggc 2653 cccaggttcg atcaactcga agccagcctc cagcatctgg ccttccacaa gcttagagcg 2713 agggRtgcta tgtgagctaa gtagtaggca attagctgag gggccccagg Nggaaccagt 2773 ggtggaaggg attNcggctt ggggttgNcc agtgggtttg Ncccagttgg ggggaggcgc 2833 aaccagtggt ggaagggatt ccgcctgggc tgcccagtgg tgtgcccagt cgtggaggcg 2893 cagtgcccag gtgaggagat caccatgttg ggcagcctga cagtccctgc gtctataagg 2953 aaggaagcca atgcctgcct ttt ctccggt agtttttccc tttgatggca catctccact 3013 ttatcatagc agtgctggtg tgctcagaga atgtcttacc agtgtttcca gcttcgggtg 3073 acatttaaaa agtcccaatt atctcagctt gggctacagt aatgtggccc tgatgaggga 3133 ccaggttctg agaaggagcc ccttgagtgg ccagtcaagg aggccagttt tcctcctagt 3193 ctatgatgat tgcaagagga ttcccggttg ttggggaaga ctaccgaggg cacacaccag 3253 ctcagaactg tttgttgggg acatttcatg acatgttcct tggaggtgag tctcacacgg 3313 gaatggtttc cagagctttt ctcctggact gggtacatat ataccatgcc ccttgggaag 3373 cttggataga aggatacttg ccaccgcctg tgagatattt tgaggtactg atgtctgaga 3433 cctgcctccc atgtcctaga ctcaccccac ccttctggcc taggtatagg gagaactggg 3493 gctccagacc cagtctccaa gtcactacca aaattcccca gctgcggtcc agttccttct 3553 gccactgtgg atgcagctca ggaaggcctc agggcagtag gccaggcttt ccctgctaca 3613 accataccct gctgaggcta tcagtggggt tgctgggaga agacatgaat ctcctgagct 3673 agagatggtt ccggaagtag gtaatctggg gcccggtatc acccactagc tcccacactg 3733 ttgttgctga tagacgctta tgaatgcctt caacacaacc caaagacccg gacacagtaa 3793 caggtcacct gctggcttcc gagtcctct t gtgttcagtg tccagactcc ttccatgtat 3853 tctgttaaga aaccacatac agccagaagg cccatggcca ggtcctgggg actgattaaa 3913 tatcctgagg tggaggggct cacataagcc cagcacagca ctgaagcccc tgccttaggt 3973 tcctttctgt ttgggcaagg gggggcatca gtgcagctgc gaaggccctt ccaagccata 4033 atgacaggag tgtagaagcc cagaagggtg gtgatagggc tgtctccagt gagctcatct 4093 tctcagcagg tatactcaaa acagcacact gggaggacaa acacaggcct ccgctaagca 4153 tttaatagca gggaaataaa ggagctggag atgttctaac tgggcctcgc ttcagcgttc 4213 aggcatccat ctgtatgtcc atgtgccagg gctgctcttc ttcccagcaa tagttcctgg 4273 aatcagctgc ctgggctttt ctagactgaa tctgattcca ccatctatct tcgtgactgg 4333 cccttgagcc aggagttact gctgtgtgac cagtgggaga tagctacctt ctctgggctc 4393 ctagcagcat aggagtgagg agcgccccta atactagctc actgcaacat gtggtaggcc 4453 cacacacgac cacctcctct tccctcacca gtgtggcatc tccatagtac cctacctcgg 4513 ctgtgcctca tcatggctgc cagggatggc ccacaagtat gagctcaact ctgggacctc 4573 atggccaggg attctgggat acttctcagc atggtgggag atatttcttt gaacaagtgg 4633 gaaaggattt ggagctagct acaaggatat ctgg atggga catcctgaat ggatctatcc 4693 ccagtttggt gaccctgaga gtcatgtgac accctcaagg gccactgtct ggtgccagtg 4753 aggccctggc tctaacacat tcagagaggc tcagatttag gtctctttat ccctgtggtt 4813 gacttcctga gctgttccag atgtctagca catcaggtgt gccccagtgc caaagaccct 4873 aggtgacatg ttttaaagtt tgcttgaacc ttattgcaaa tgtatttgaa gtgagggtct 4933 tccacataaa aattgggatg gaccccaggg ggcttggtcc ggccctttct actccctacc 4993 cctatgcaca tacatataaa tatatataca tataaataca tatatataaa catacatata 5053 catataagca tgcatatgtg tatatataca tacacataca tataaacata catgtacata 5113 cacatataca catacatata tacacatata cacatacata taaatataca tatacacata 5173 catataaaca taaatataca tatacatata catcatatac atatatacat acacatatac 5233 acatgcatat atacatacaa taaatatacc tatacatcat atacatacac atatacatac 5293 acatataaac atacgtatat attatgtaca tatacatacc caatcttttc tgtctgtagt 5353 gcaccacttg tataagatgg gacagttcca aacctgaaga agggacagtt cttagaggtg 5413 ttaaaggcgt atgacctgtg tagctgttga ggtgtcctgc ctctctgtct cttccgtctg 5473 accttcccaa gatggtttta gacacccact tctccttaca ccggcactgc ataaagactg 5533 tggtagtatg catctgtagt tccaacattg ggaaacagag gccaggtgaa aggaatgtgt 5593 ggctatcctt acccacacag tgatatggtt aaggccagca tgggttacat gagacctgct 5653 tcagaaaaaa agaaggaaca gggaaaatgt tctccccatc aggtaaaacg catgtatgca 5713 aaggtctgag tgtgaatctc agaattcatg tagaagttgg gcatggtggc acacctggaa 5773 ccccactgac aggaaggcag gcagagaggg ccagatagcc agaaggctgc aggccaagga 5833 gcatagagta ccaggcctac gagagaacca ccccgagcaa aaagtggatt accttgagag 5893 tttgtgctct gacctctcct aatgtgttgt gtacataggc atgtgtgccc cctcacatag 5953 gtaccctcca catgaatgcg cacacacaca cacacgcaca cacaatgaga gagggacaga 6013 gacaaagaga ggtagagaca gaaaatcaga cacacacaca cacacgcgtg cacatacaca 6073 tgtgcacatg cacacacaca cactgacaca caccacacac acaatgagag agagacagag 6133 acaaagagag gtagagatag aaagacatac acagagagat agactgagtc agaggtagag 6193 acagaaagac acacacacac acacacacac acacacacac acagagacac agagagacag 6253 ggacagcaaa cgagcaactt ctttttcgtg ctggccccag attggtctat ctagaggctg 6313 agcactccct gcccacccat tgtcctttgg agccaccctg cgcca gggcg aggcaggaca 6373 gtggccagac ttcatagccc agctcccctt ctcgtcccca tggcctctga accagtgaga 6433 gatttattta tagagacaag aactagttct ctcccacctc ttacaagagg agagcttctg 6493 cttgaattag tgtttccccc cttgttcccc gctcccttgg caaagggtca cagacatttc 6553 tattacctcc aaacccactg ggtctatgaa ggggcttcgt gggaagccag gactctctgg 6613 gctttggaat cagcccgctg ttcaagttta ggccactgaa cagcaattga tctgcaggag 6673 gtggtccttg ggagaattca tggagaaaaa tccactgcta atcatgctat ttataattcc 6733 ttcatatgct tcgcgaaggg ttcaggacaa tgagaaactc agcccatcct ccccagggag 6793 gtccttgcct cagaagctca cttgtcattg tRctttgaag tccatggatt tgtgtggcat 6853 gtaactgacc gagggacctt ggcagcctta cagaagagca cggatcttgg cctcaccctc 6913 acccgtcagg cagtcagtgg gccctgcctg cagtaccacc aggactgagg actctgtctc 6973 tctttcctgt tgaaagaaat ggttgtcctg Rccccacgca gactttccta tagtccttgt 7033 aatgatctgg agcccacatc tggagtcctt gctccccacg gccaatacca taatttcttc 7093 ctttatctaa ggcaaaccac agcatccttc cctcacaggt gttaccccgt gtctgccaac 7153 taccctcctc ctttgttcgg acttagccat cgggcatgag ttgtggccca tttggagcct 7213 ctgaggtttc ctgaggtggg gaccctccgc ttttgtttac gtgtaaccta cctctgaggt 7273 gacttcctgt atactccatg ggttatagct cccagactct gctatctttg tgcagtataN 7333 ccgtgcactg gtggtataat ggtgggcaaa gctggctttc attgtgcctc attttaccac 7393 agattcccca gctcaagtca catttctgcc agacggtgta ttcaggtgaa gcagaaataa 7453 atccacttgg cggaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 7507

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of differential display PCR. In the figure, C is the healthy hypoglossal nucleus, O
Shows the results of PCR products using the injured sublingual nucleus as a template.

FIG. 2 is a diagram showing the amino acid sequence of the DD13 protein (derived from rat) of the present invention in one-letter title.

FIG. 3 is a diagram showing a hydrophobicity plot of the amino acid sequence of the DD13 protein (derived from rat) of the present invention.

FIG. 4 is a diagram showing the results of Northern hybridization. In the figure, A shows the results of hybridization with the probe derived from the differential display fragment, B shows the results of hybridization with the probe corresponding to the ORF of DD13 (C shows healthy hypoglossal nucleus, O shows damaged hypoglossal nerve) Nucleus).

FIG. 5 shows the results of Western blotting of a membrane fraction prepared from cells transfected with the DD13 gene. In the figure, DD13 indicates a membrane fraction prepared from cells transfected with the DD13 gene,
mock indicates the results of cells transfected with the vector alone.

FIG. 6 shows the results of in situ hybridization. In the figure, (a) shows the results after sublingual axotomy, and (b) shows the results after ischemia.

FIG. 7 shows the results of in situ hybridization. In the figure, (a) shows the embryonic period (embryonic day 1).
7) shows the results of the nervous system, and (b) shows the results of intestinal ganglion neurons.

FIG. 8 is a view showing the results of an RNase protection assay. The numbers in the lanes in the figure are 1 (brain), 2 (liver), 3 (kidney), 4 (thymus), 5 (muscle), 6 (lung),
7 (heart), 8 (normal sublingual nucleus), 9 (postoperative sublingual nucleus), 10
(Embryonic day12 whole body), 11 (embryonic day 16
Brain), 12 (1 day old brain).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) (C12N 9/64 C12R 1:91) F term (reference) 4B024 AA01 BA14 BA61 CA04 4B050 CC03 DD11 LL01 4B064 AG01 AG26 CA09 CA19 DA03 4C084 AA01 AA07 BA01 BA22 BA44 DA39 DC09 NA05 NA14 ZA011 ZC192

Claims (8)

[Claims] 1. A protein specified by the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and having a metalloendoprotease activity. 3. A polynucleotide encoding the protein according to claim 1 or 2. 4. The polynucleotide according to claim 3, which is a DNA comprising a nucleotide sequence from A at position 96 to G at position 2420 in the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing. 5. A DNA comprising 12 or more consecutive bases in the base sequence shown in SEQ ID NO: 2 in the sequence listing. 6. An antibody that recognizes the protein according to claim 1 or 2. 7. A metalloendoprotease comprising the protein according to claim 1 or 2. 8. A medicament comprising the protein according to claim 1 or 2 as an active ingredient.