JP2002000274A - Novel genes and proteins encoded by them - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel protein HUCEP- derived from human cerebral cortex.
9 and the gene encoding it, husep-9. The novel protein HUCEP- is cloned by cloning from a cDNA library derived from human cerebral cortex.
Thus, a novel protein, HUCEP-9, is obtained by culturing a transformant with an expression vector having the gene, hucep-9, which encodes Hucep-9.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a protein associated with Parkinson's disease, a DNA encoding the protein, and an antibody useful for detecting Parkinson's disease.

[0002]

2. Description of the Related Art Parkinson's disease is one of the neurodegenerative diseases mainly caused by dyskinesia such as trembling of hands and feet. It is said that there are about 100,000 patients in Japan and more than 500,000 in the United States. I have. In 1997, a causative gene for familial Parkinson's disease was identified and found to be none other than α-synuclein, previously known as a synaptic protein (Polymeropoulos R et al Science 276, 2045 (1945
97)).

[0003] That is, it has been clarified that a single nucleotide mutation occurs in the gene encoding α-synuclein, and that a mutation of one amino acid is introduced into α-synuclein protein, thereby causing Parkinson's disease. α-synuclein is a protein consisting of 140 residues present in presynapses (Maroteaux L et al
J Neurosci 8, 2804 (1988)) showed that antibodies against α-synuclein react with inclusions in substantia nigra neurons called Lewy bodies, a pathological feature of Parkinson's disease (Spillantini). MG et al Nature388, 839
(1997); Wakabayashi K et al Neurosci Letters 239: 4
5 (1997)).

It has also been found that α-synuclein itself is aggregated in Lewy bodies (Baba M et al Am
J Pathol 152, 879 (1998)). These facts revealed that at least α-synuclein is a component of Lewy bodies, but even if α-synuclein alone is overexpressed in cells, Lewy bodies are not formed. Also, α-synuc
synphilin-1 was cloned as one of the proteins interacting with lein (Engelender S et al Nat Genet 22,
110 (1999)). Anti-synphilin-1 antibody also reacts with Lewy bodies (Wakabayashi K et al Annals Neurol 47,521 (2
000)) strongly suggested that proteins other than α-synuclein are deeply involved in Lewy body formation.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel protein present in Lewy bodies, a gene encoding the protein, and an antibody against the protein.

Another object of the present invention is to provide a screening method for finding a compound that regulates the expression of the protein or a compound that inhibits the aggregation of the protein.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies on a protein encoded by a gene specifically expressed in human brain tissue and found that a novel protein (hereinafter referred to as HUCEP) was obtained.
-9) and the gene encoding it (hereinafter, huc)
ep-9) was successfully isolated. Furthermore, it was found that HUCEP-9 has guanine nucleotide exchange factor activity. When an antibody against HUCEP-9 was prepared, it was found that the antibody reacts with Lewy bodies, which is a pathological feature of Parkinson's disease. It was completed.

That is, the present invention relates to (1) (a) SEQ ID NO:
Novel protein HUCEP consisting of the amino acid sequence of 1
-9 or (b) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and having a guanine nucleotide exchange factor activity; (2) a sequence; A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of No. 1, and constituting Lewy bodies, (3) SEQ ID NO: 3
A peptide comprising the amino acid sequence of any one of claims 1 to 5,
(4) a gene encoding the protein represented by (1) or (2), (5) (c) a DNA comprising the nucleotide sequence of SEQ ID NO: 2, or (d) a DNA of SEQ ID NO: 2.
And stringent conditions, and
DNA encoding a protein having a guanine nucleotide exchange factor activity, (6) a DNA that hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and constitutes a Lewy body, (7) SEQ ID NO: : DNA containing the nucleotide sequence of (6), (8) an antibody against the protein of (1) or (2), (9) against an oligopeptide having a part of the protein of (1) or (2). (10) A method for screening for a Parkinson's disease test agent containing the antibody according to (8) or (9), or a compound or a salt thereof that inhibits aggregation of the protein according to (1) or (2). , (12) (4)-(6)
(13) a method for screening a compound or a salt thereof, which promotes or inhibits the expression of the gene according to any of
A compound or a salt thereof obtained by using the screening method according to (12) or (13).

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The gene hucep-9 can be isolated as a cDNA fragment containing the gene from a cDNA library derived from human cerebral cortex. The cDNA library used by the present inventors was prepared based on the human cerebral cortex mRNA commercially available from Clonetech, but the human cerebral cortex mRNA commercially available from Stratagene was used. In any case, cDNA can be similarly prepared.

In the above-mentioned cDNA library, as a method for identifying a cDNA which seems to have a gene specifically expressed in human brain tissue, the method of Okubo et al.
kubo et al., Nature Genet., 2, 173 (1992)) can be used to analyze the frequency of appearance of gene expression. Specifically, using mRNA of human cerebral cortex as a template,
A vector plasmid opened with an appropriate restriction enzyme to which oligo dT was bound at one end was used as a primer for cD
After performing NA synthesis, restriction enzyme MboI and restriction enzyme Ba
Cut with mHI. Since this vector was prepared using dam methylase-positive Escherichia coli as a host, the A residue of “GATC”, which is a recognition sequence of MboI, is methylated. Therefore, MboI cuts only the newly synthesized cDNA portion. This vector has one BamHI cleavage site near the end opposite to the end to which oligo dT is bound.
Since this enzyme has only one site, this enzyme cleaves the vector at one site, and further adds B to the newly synthesized cDNA portion.
If the amHI recognition sequence is present, the site is also cleaved.
BamHI and MboI are composed of the sequence "GATC" and generate the same cohesive end. After cutting with both enzymes, the DNA can be ligated with DNA ligase to close the plasmid.

In the present method, a cDNA library was constructed by transforming Escherichia coli using the plasmid thus prepared. Therefore, the library starts from the poly-A site at the 3 ′ end of each mRNA and its 5 ′ end.
It includes the region up to the site where the base sequence GATC first appears in the side portion. An appropriate number of recombinants are randomly selected from the cDNA library, and the cDNA in each recombinant is extracted to determine the entire base sequence. The present method is used to determine whether a cD having a specific sequence determined in this way
This is a method for discriminating an organ-specific gene and a highly expressed gene based on how many NA fragments are identified from randomly selected recombinants. In this method, extraction of the recombinant cDNA and determination of the nucleotide sequence of the cDNA are all performed by various operation methods known to those skilled in the art (Molecular Cloning, 2nd ed., Cold Spring Harbor La.
b.Press, (1989), and other methods described in technical manuals that introduce standard methods for those skilled in the art.
Can be performed.

In the method for identifying a highly expressed gene, the total number of recombinants selected at random is suitably several hundreds to about 1,000, but if necessary, more recombinants may be processed. . We performed the above method and found that 77
The nucleotide sequences of all the cDNA fragments in the 0 recombinants were determined, and from those, the cDNA fragments whose frequency of appearance as cDNA having the same sequence was 2/770 or more were determined.
DNA fragments having genes specifically expressed in the human brain were selected as candidates.

[0013] As described above, the above cDNA fragment is composed of mR
It contains only a part of the 3 'end of NA. Therefore, the present inventors obtained a full-length cDNA based on the nucleotide sequence information of the region (hereinafter, 3 ′ fragment).

This is based on the use of a human cerebral cortex cDNA library commercially available from Clontech as a template, and an oligonucleotide of an appropriate length having a sequence in the above 3 ′ fragment and a comparable length having a sequence in a vector. Each oligonucleotide was synthesized and used as a primer, thereby performing the PCR method. As a result, a DNA fragment of about 2.6 kb could be amplified. In this case, a human cerebral cortex cDNA library commercially available from Stratagene can be used as a template. This is also the mR of the human cerebral cortex, commercially available from Clonetech or Stratagene.
It can also be performed using NA as a template and using a 5 'RACE kit from Clonetech or Gibco. Further, this can also be performed by screening the above human cerebral cortex cDNA library by colony hybridization or plaque hybridization using the above 3 ′ fragment as a probe according to a conventional method.

The cDNA fragment amplified by the above method is commercially available from Novagen pT7Blue.
It was incorporated into a T-vector and the entire nucleotide sequence was determined according to a conventional method. At this time, the sequence was confirmed by independently obtaining two clones of the recombinant DNA and determining the nucleotide sequence of each cDNA fragment.

To confirm that the gene considered to be present in the cDNA fragment selected by the above method is specifically expressed in brain tissue, the organ-specific expression frequency of the cDNA sequence was confirmed by Northern hybridization. It can be done by doing. Specifically, cDNA fragments, which are commercially available from Clonetech or Stratagene, are extracted from human organs, fractionated by agarose gel electrophoresis, transferred to a membrane filter, and then selected by the above method. Using as a probe, hybridization was carried out according to a conventional method. We used this method to examine organ specificity for expression of the cDNA sequence. As a result, it was confirmed that although the cDNA sequence was somewhat expressed in organs, organs, cells, etc. other than the brain, it was specifically expressed in the cerebral cortex.

This strongly suggests that the cDNA sequence contains a desired gene which is specifically expressed in the human brain and essential for maintaining normal brain function.

A region encoding a protein in the base sequence (O
RF, open reading frame) can be confirmed by a general-purpose method of analyzing a nucleotide sequence using a computer program. The present inventors who were convinced of the existence of the target gene in the cDNA sequence found an ORF in the sequence using a computer, and identified this gene as a gene hucep-9 (abbreviation for human cerebral protein). The protein encoded by the gene was converted to HUCEP-9.
It was named.

The gene husep-9 is a gene consisting of 2244 base pairs (bp) shown in SEQ ID NO: 2. Using this gene hucep-9, a recombinant gene can be prepared by a general gene recombination technique using an appropriate host vector system. Suitable vectors include plasmids derived from E. coli (eg, pBR32
2, pUC118, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pC194, etc.), yeast-derived plasmids (eg, pSH19, etc.), and animal viruses such as bacteriophages, retroviruses, and vaccinia viruses. Upon recombination, a translation initiation codon and a translation termination codon can be added using an appropriate synthetic DNA adapter. In order to further express the gene, an appropriate expression promoter is connected upstream of the gene. The promoter to be used may be appropriately selected according to the host. For example, when the host is Escherichia coli, a T7 promoter, a lac promoter, a trp promoter, a λPL promoter, etc.
When the host is a bacterium belonging to the genus Bacillus, an SPO-based promoter or the like is used. When the host is yeast, a PHO5 promoter, a GAP promoter, an ADH promoter, or the like is used. A promoter or the like can be used respectively.

The gene can also be expressed as a fusion protein with another protein (eg, glutathione S-transferase, protein A, etc.). The fused HUCEP-9 thus expressed can be excised using a suitable protease (eg, thrombin, enterokinase, etc.).

Hosts that can be used for the expression of hucep-9 include Escherichia col.
Various strains of i , various strains of Bacillus subtilis belonging to the genus Bacillus , various strains of Saccharomyces cerevisiae as yeast, COS-7 cells and CHO cells as animal cells can be used.

As a method for transforming a host cell using the above-mentioned recombinant vector, a conventional method or a transformation method generally used for each host cell can be applied.

The novel protein HUCEP-9 of the present invention
Has all amino acids 747, as shown in SEQ ID NO: 1.
It is a protein having a molecular weight of 83921.2 daltons consisting of residues. In addition, it was found that Ras guanine nucleotide exchange factor motifs exist at the 242-297th position and the 463-672th position in the amino acid sequence.

In the present invention, in addition to the DNA sequence shown in SEQ ID NO: 2, the DNA hybridizes with the DNA,
In addition, a DNA encoding a protein having guanine nucleotide exchange factor activity is also within the scope of the present invention.

The guanine nucleotide exchange factor activity is
It refers to the action of removing guanosine diphosphate from a protein that can bind to guanosine diphosphate or guanosine triphosphate and exchanging it for guanosine triphosphate.

A DNA that hybridizes with the DNA shown in SEQ ID NO: 2 and encodes a protein constituting a Lewy body is also within the scope of the present invention.

That is, in the full-length sequence of the gene hucep-9, various artificial treatments such as site-directed mutagenesis, random mutation by treatment with a mutagen, mutation / deletion / ligation of a DNA fragment by restriction enzyme cleavage, etc. Even if the DNA sequence is partially changed, these DNA mutants hybridize with the gene hucep-9 under stringent conditions, and antibodies against the protein encoded by the DNA mutant are used as described below. As long as it has reactivity with the body, it is within the scope of the present invention regardless of the difference from the DNA sequence shown in SEQ ID NO: 2.

The degree of the DNA mutation is determined by the gene huc
If it has a homology of 90% or more with the DNA sequence of ep-9, it is within the allowable range. Also, the gene husep
As a degree of hybridization with -9, when a probe is labeled under normal conditions (eg, DIG DNA Labeling kit (Boehringer Mannheim Cat No. 1175033)), a 32 ° C. DIG Easy Hyb solution (Boehringer Mannheim) (Cat No. 1603558), and 0.5 × SSC solution (0.1% [w / v]) at 50 ° C.
Conditions for washing the membrane in SDS (including SDS) (1 × S
(SC is 0.15 M NaCl and 0.015 M sodium citrate), as long as it hybridizes to the gene hucep-9 by Southern hybridization.

Further, as described above, the gene hucep-9 was used.
A protein encoded by a mutant gene having a high homology with the protein having a guanine nucleotide exchange factor activity, or an antibody against the protein having reactivity with Lewy bodies is also within the scope of the present invention. Things.

That is, even a mutant in which one or more amino acids of the amino acid sequence of the novel protein HUCEP-9 is deleted, substituted or added, has a guanine nucleotide exchange factor activity, or If the antibody reacts with Lewy bodies, the variants are within the scope of the invention.

The side chains of amino acids which are constituents of a protein are different from each other in hydrophobicity, charge, size, etc., but substantially affect the three-dimensional structure (also referred to as a three-dimensional structure) of the whole protein. Some conservative relationships in the sense that they are not are known empirically and by physicochemical measurements. For example, for substitution of amino acid residues, glycine (Gly), proline (Pro), Gl
y and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine (C
ys) and threonine (Thr), Thr and serine (Se)
r) or Ala, lysine (Lys) and arginine (A
rg), and the like.

Therefore, the protein HU shown in SEQ ID NO: 1
Even for a mutant protein due to substitution, insertion, deletion, etc. on the amino acid sequence of CEP-9, the mutation is a highly conserved mutation in the three-dimensional structure of HUCEP-9 protein,
If the mutant protein has the same guanine nucleotide exchange factor activity or antigenicity as HUCEP-9, these can be said to be within the scope of the present invention. The degree of mutation is within a range in which homology to the amino acid sequence shown in SEQ ID NO: 1 is 90% or more.

The present invention also relates to the protein HUCEP of the present invention.
A method for detecting Parkinson's disease, comprising using an antibody against -9. Specifically, the present invention relates to HUCE
An oligopeptide having a part of the sequence of P-9 or HUCEP-9 is administered as an antigen to animals such as rabbits, goats, donkeys and mice, and an antiserum or monoclonal antibody against the antigen is obtained using a known method. A method for immunohistochemically staining a pathological specimen using the antibody to detect whether or not the patient who provided the specimen has Parkinson's disease. An oligopeptide having a part of the sequence of HUCEP-9 is, for example, Arg Ala Glu Gly Asn Pr
o Arg Gly Thr Asp Leu Glu Asn Pro Arg Glu (SEQ ID NO: 3), Met Ala Arg Thr Ser Ser Arg Ala Pro Cys
(SEQ ID NO: 4), Asp Lys Leu Arg Arg Met Lys Ala Th
r Phe Gln (SEQ ID NO: 5) can be mentioned.

Furthermore, the present invention is a method for screening a compound or a salt thereof that inhibits HUCEP-9 aggregation.

Furthermore, the present invention is a method for screening a compound or a salt thereof that promotes or inhibits expression of hucep-9. For example, when a test compound is brought into contact with a cell into which the present gene and its upstream region have been incorporated and the amount of mRNA complementary to the present gene is increased as compared with the case where the test compound is not contacted, a substance that promotes the expression of the present gene If the amount of the mRNA is reduced, it can be selected as a substance that inhibits the expression of the present gene.

[0036]

HUCEP-9 is present in Lewy bodies and has guanine nucleotide exchange factor activity.
It is thought to be related to Parkinson's disease. Therefore, researching HUCEP-9 is expected to be useful as a new therapeutic agent for cranial nerve diseases.

By preparing an antibody that reacts with HUCEP-9, a Parkinson's disease test can be performed.

Hereinafter, the present invention will be described in detail with reference to Examples, but it goes without saying that the present invention is not limited to these Examples.

[0039]

EXAMPLES Example 1 Cloning of gene husep-9 1) Determination of partial sequence of gene essential for maintaining normal function of cerebrum Using mRNA of human cerebral cortex (Clontech) as a template, the method of Okubo et al. et al. Nature Genet., 2,
173 (1992)), a cDNA library of the cerebral cortex was prepared.

Next, 7
Seventy recombinants were selected and used in a conventional method (Molecular Cloning,
According to 2nd. Ed., Cold Spring Harbor Lab. Press, 1989, the same applies hereinafter), the recombinant DNA was extracted, and the nucleotide sequence on the 3 ′ side of the cDNA portion was determined. For sequencing, use a DNA sequencer manufactured by PE Applied Biosystems (AB
I PRISM377) and its reaction kit. As a result of analyzing the expression frequency of each DNA fragment in the 770 recombinants, the expression frequency of the gene having the sequence shown in FIG. 1 (sequence-1: SEQ ID NO: 7) was 2/770. 2) Amplification of cDNA fragment containing sequence-1 cDNA fragment containing sequence-1 (SEQ ID NO: 7) was amplified by the following method.

First, an oligonucleotide having a sequence complementary to a part of sequence-1 (FIG. 1; sequence-2 (SEQ ID NO: 8)) was ligated to DN (manufactured by PE Applied Biosystems).
A was synthesized using an A synthesizer (ABI 380B). Next, an oligonucleotide having a sequence near the cDNA insertion site of the lambda phage cloning vector (λDR2) (sequence-
3: SEQ ID NO: 9) was synthesized in the same manner. Human Brain cerebral cortex using λDR2 as a cloning vector
PCR was performed using 5′-STRETCH cDNA library (manufactured by Clonetech Laboratories) as a template and an oligonucleotide of sequence-2 (SEQ ID NO: 8) and an oligonucleotide of sequence-3 (SEQ ID NO: 9) as primers. The reaction was performed using a kit manufactured by Takara Shuzo Co., Ltd. (Takara LA PCR Kit Ve
Using r.2), PCR Thermal Cycler MP manufactured by Takara Shuzo Co., Ltd. was used.

Reaction composition cDNA library (10 ⁸ pfu / ml) 5 μl 10 × PCR buffer 5 μl 2.5 mM dNTP 1 μl 10 μM oriconucleotide (sequence-2) 2 μl 10 μM oriconucleotide (sequence-3) 2 μl water 34.5 μl LA DNA polymerase 0.5 μl Total volume 50 μl Reaction conditions After holding at 95 ° C. for 2 minutes, react at 95 ° C. for 20 seconds,
The solution was cooled to -1C at a rate of -1C / 2 seconds, kept at 58C for 1 minute, and further kept at 72C for 4 minutes. This was repeated 35 times to amplify the target sequence.

According to the above method, a DNA fragment (about 2.6 kb) having a part of sequence-1 was specifically amplified (FIGS. 2 and 3). 3) Subcloning into base sequence determination vector The DNA amplified in 2) was fractionated by agarose gel electrophoresis (gel concentration 1%) according to a conventional method. The gel was stained with ethidyl bromide, and then irradiated with ultraviolet light to cut out a gel containing a target band.

The extraction and purification of the DNA fragment from the agarose gel was performed using Geneclean II Kit (manufactured by Bio 101).

This purified DNA fragment was subcloned into a base sequence determination vector, pT7Blue T-Vector (Novagen). The ligation solution was prepared using Takara Shuzo Co., Ltd. kit (Takara DNA Ligation Kit Ver.2).
The reaction was performed at 1.5 ° C. for 1.5 hours.

Using the above reaction solution, Escherichia coli K12 strain DH5 was transformed according to a conventional method.

Transformants were treated with Ampicillin (Amp) 50
μg / ml, 5-Bromo-4-Chloro-3-indolyl-β-D-galact
oside (X-gal) 40 μg / ml, Isopropyl-β-DT
The cells were plated on LB agar medium containing 100 μM of hio-Galactopyranoside (IPTG) and cultured at 37 ° C. overnight.

The white colony was inoculated into 10 ml of an LB liquid medium containing 50 μg / ml of Amp and cultured overnight at 37 ° C., and the cells were collected by centrifugation.
Recombinant DNA with lasmid Miniprep Kit (Qiagen)
Was purified. The recombinant DNA thus constructed is
It was named Thusep-9. 4) Determination of base sequence of DNA fragment For base sequence determination, a DNA sequencer manufactured by PE Applied Biosystems was used, and a dye terminator method was used. Oligonucleotides were synthesized based on the determined base sequence, and the entire base sequence was determined by the primer walking method. The nucleotide sequences of both strands were determined. FIG. 3 shows the entire nucleotide sequence of the cDNA of the clone. Since the base sequence contained the upstream of the complementary strand of sequence-2, the target gene (human cerebral protein-9, huce
p-9) was confirmed to be cloned.

The cDNA was translated into a translation region (open readi) encoding a protein (HUCEP-9) consisting of 747 residues.
ng frame, ORF). Example 2 Preparation of Anti-HUCEP-9 Antibody An oligopeptide having a part of the sequence of HUCEP-9 (FIG. 1; sequence-4 (SEQ ID NO: 3)) was synthesized using a peptide synthesizer manufactured by Millipore. At that time, C
A peptide to which a ys residue is added is synthesized, and KLH (Keyhole Limpet Hem) is synthesized through the Cys residue using a known method.
Rabbit immunized rabbits to obtain antiserum. After immunization, blood was collected over time to prepare serum. The peptide used as the antigen was fixed on a 96-well microtiter plate, and the antibody titer in the serum was measured by ELISA (FIG. 4). After confirming that the antibody titer was sufficiently increased, whole blood was collected, and the IgG fraction was purified from the serum by protein A column chromatography. Next, an antigen-specific IgG fraction was purified by affinity chromatography using a column to which the antigen was bound. The antibody titer of the fraction was measured by ELISA (FIG. 5). Example 3 Immunohistological Staining Using Anti-HUCEP-9 Antibody Using the IgG prepared in Example 2 and immunostaining of human brain tissue using a known method, one of the pathological features of Parkinson's disease was determined. One Levy body dyed strongly (Fig. 6).

[Sequence List] <110> TAISHO PHARMACEUTICAL Co. Ltd. <120> Novel Gene and Protein Ecoded <130> 00TS-P3061 <141> 2000-06-23 <160> 2 <210> 1 <211> 747 <212 > PRT <213> HOMO SAPIENS <400> 1 Met Ala Arg Thr Ser Ser Arg Ala Pro Cys Ser Pro Thr Ser Val 5 10 15 Ser Asp Val Asp Ser Asp Ala Leu Ser Arg Gly Asn Phe Glu Val 20 25 30 Gly Phe Arg Pro Gln Arg Ser Val Lys Ala Glu Arg Ala Gln Gln 35 40 45 Pro Glu Ala Gly Glu Asp Arg Arg Pro Ala Gly Gly Ala Ser Asp 50 55 60 Val Glu Ala Val Thr Arg Leu Ala Arg Ser Lys Gly Val Gly Pro 65 70 75 Ala Leu Ser Pro Gly Pro Ala Gly Phe Gln Ser Cys Ser Pro Gly 80 85 90 Trp Cys Ser Ala Phe Tyr Glu Ala Asp Cys Phe Gly Ala Asp Val 95 100 105 His Asn Tyr Val Lys Asp Leu Gly Arg Gln Gln Ala Asp Gly Ala 110 115 120 Leu Pro Asp Ala Gln Ser Pro Glu Leu Glu Gln Gln Leu Met Met 125 130 135 Glu Lys Arg Asn Tyr Arg Lys Thr Leu Lys Phe Tyr Gln Lys Leu 140 145 150 Leu Gln Lys Glu Lys Arg Asn Lys Gly Ser Asp Val Lys Thr Met 155 160 165 Leu Ser Lys Leu Lys Gly Gln Leu Glu Glu Met Lys Ser Arg Val 170 175 180 Gln Phe Leu Ser Leu Val Lys Lys Tyr Leu Gln Val Met Tyr Ala 185 190 195 Glu Arg Trp Gly Leu Glu Pro Cys Thr Leu Pro Val Ile Val Asn 200 205 210 Ile Ala Ala Ala Pro Cys Asp Thr Leu Asp Phe Ser Pro Leu Asp 215 220 225 Glu Ser Ser Ser Leu Ile Phe Tyr Asn Val Asn Lys His Pro Gly 230 235 240 Gly Arg Gln Lys Ala Arg Ile Leu Gln Ala Gly Thr Pro Leu Gly 245 250 255 Leu Met Ala Tyr Leu Tyr Ser Ser Asp Ala Phe Leu Glu Gly Tyr 260 265 270 Val Gln Gln Phe Leu Tyr Thr Phe Arg Tyr Phe Cys Thr Pro His 275 280 285 Asp Phe Leu His Phe Leu Leu Asp Arg Ile Asn Ser Thr Leu Thr 290 295 300 Arg Ala His Gln Asp Pro Thr Ser Thr Phe Thr Lys Ile Tyr Arg 305 310 315 Arg Ser Leu Cys Val Leu Gln Ala Trp Val Glu Asp Cys Tyr Ala 320 325 330 Val Asp Phe Pro Arg Asn Ser Gly Leu Leu Gly Lys Leu Glu Asp 335 340 345 Phe Ile Ser Ser Lys Ile Leu Pro Leu Asp Gly Ser Ala Lys His 350 355 360 Leu Leu Gly Leu Leu Glu Val Gly Met Asp Arg Arg Ala Glu Gly 365 370 375 Asn Pro Arg Gly Thr Asp Leu Glu As n Pro Arg Glu Ala Glu Glu 380 385 390 Asp Ala Arg Pro Phe Asn Ala Leu Cys Lys Arg Leu Ser Glu Asp 395 400 405 Gly Ile Ser Arg Lys Ser Phe Pro Trp Arg Leu Pro Arg Gly Asn 410 415 420 Gly Leu Val Leu Pro Pro His Lys Glu Arg Pro Tyr Thr Ile Ala 425 430 435 Ala Ala Leu Pro Lys Pro Cys Phe Leu Glu Asp Phe Tyr Gly Pro 440 445 450 Cys Ala Lys Thr Ser Glu Lys Gly Pro Tyr Phe Leu Thr Glu Tyr 455 460 465 Ser Thr His Gln Leu Phe Ser Gln Leu Thr Leu Leu Gln Gln Glu 470 475 480 Leu Phe Gln Lys Cys His Pro Val His Phe Leu Asn Ser Arg Ala 485 490 495 Leu Gly Val Met Asp Lys Ser Thr Ala Ile Pro Lys Ala Ser Ser 500 505 510 Ser Glu Ser Leu Ser Ala Lys Thr Cys Ser Leu Phe Leu Pro Asn 515 520 525 Tyr Val Gln Asp Lys Tyr Leu Leu Gln Leu Leu Arg Asn Ala Asp 530 535 540 540 Asp Val Ser Thr Trp Val Ala Ala Glu Ile Val Thr Ser His Thr 545 550 555 Ser Lys Leu Gln Val Asn Leu Leu Ser Lys Phe Leu Leu Ile Ala 560 565 570 Lys Ser Cys Tyr Glu Gln Arg Asn Phe Ala Thr Ala Met Gln Ile 575 580 585 585 Leu Ser Gly Leu Glu His Le u Ala Val Arg Gln Ser Pro Ala Trp 590 595 600 Arg Ile Leu Pro Ala Lys Ile Ala Glu Val Met Glu Glu Leu Lys 605 610 615 Ala Val Glu Val Phe Leu Lys Ser Asp Ser Leu Cys Leu Met Glu 620 625 630 Gly Arg Arg Phe Arg Ala Gln Pro Thr Leu Pro Ser Ala His Leu 635 640 645 Leu Ala Met His Ile Gln Gln Leu Glu Thr Gly Gly Phe Thr Met 650 655 660 Thr Asn Gly Ala His Arg Trp Ser Lys Leu Arg Asn Ile Ala Lys 665 670 675 Val Val Ser Gln Val His Ala Phe Gln Glu Asn Pro Tyr Thr Phe 680 685 690 Ser Pro Asp Pro Lys Leu Gln Ser Tyr Leu Lys Gln Arg Ile Ala 695 700 705 Arg Phe Ser Gly Ala Asp Ile Ser Thr Leu Ala Ala Asp Ser Arg 710 715 720 Ala Asn Phe His Gln Val Ser Ser Glu Lys His Ser Arg Lys Ile 725 730 735 Gln Asp Lys Leu Arg Arg Met Lys Ala Thr Phe Gln 740 745 747 <210> 2 <211> 2244 <212> DNA <213> HOMO SAPIENS <400> 2 atggccagga ccagcagcag ggccccctgc tcacccacct cggtgtcgga tgtggactcg 60 gacgcactgt cacggggaaa cttcgaggtg gggtttcggc ctcagaggtc cgtaaaagccc gagagagccc aggagagccc gagagagccc ggcctcagac 180 gtggaggcag tgacccgact ggccaggtcc aaaggggtcg gcccagcctt gtcccccggc 240 ccagccggat tccagagctg cagccccggc tggtgcagcg ccttctacga ggccgactgc 300 ttcggggccg acgtccacaa ctacgtgaag gacctggggc ggcagcaggc ggacggggcc 360 ctgcccgacg cccagagccc ggagctggaa cagcagctca tgatggagaa aagaaactac 420 cgcaagaccc tgaagttcta ccagaaactc ttacagaagg aaaagaggaa caaaggttcc 480 gacgtcaaga ccatgctgtc caagctgaaa gggcagctag aagaaatgaa atccagggtg 540 caattcctca gcttggtcaa gaagtatctg caggtcatgt acgcggaacg ctggggcctg 600 gagccctgca ccctcccagt gatcgtgaac atcgcggccg caccctgcga cacgctggac 660 ttcagccccc tggacgagtc ctcctcgctc atcttctaca acgtcaacaa gcacccgggc 720 ggccggcaga aggcccgcat cctgcaggcc ggcacgccgc tggggctcat ggcctacctg 780 tactccagtg atgccttcct ggagggttat gtgcagcaat tcctctacac cttccgctac 840 ttctgcacac cccacgactt cctgcacttc ctcctcgacc gcatcaacag cacgctgacc 900 agggcccacc aggaccccac ctcgaccttc accaagatct acaggcggag cctctgcgtc 960 ctgcaggcct gggtggagga ctgctacgct gtggacttcc ctcggaacag cgggctgctg 1020 ggga agctag aggacttcat ctcctccaag atcctacccc tggacggctc tgccaagcac 1080 ctgctgggcc tcctggaggt gggcatggac cggcgggccg agggcaaccc tcgcggcaca 1140 gacctggaga accccaggga ggccgaggag gatgccagac ccttcaacgc cctctgtaag 1200 aggctctcag aggacggcat ctccaggaag agcttcccct ggaggctgcc ccgaggcaac 1260 gggctggtgc tgccgccaca caaggagcgc ccctacacca ttgctgccgc cctgcccaag 1320 ccctgcttcc tcgaggactt ctacggcccc tgcgccaaga ccagtgagaa ggggccctac 1380 ttcctgacgg agtacagcac tcaccagctc ttcagccagc tcacgctgct acagcaggag 1440 ttgtttcaaa agtgccaccc ggtccacttc ctgaactcac gggccctggg cgtcatggac 1500 aagagcactg ccatccccaa agccagctct tctgagtctc tttcggccaa aacctgcagc 1560 ttatttctgc ccaattacgt tcaggacaag tatctgttac agcttctaag aaacgcagat 1620 gacgtcagca cctgggtggc tgcagagatt gtgaccagcc acacctccaa gctgcaggtg 1680 aacttgctgt ccaaattttt gctgattgca aaatcttgct atgagcagag aaacttcgcg 1740 acagccatgc agatcctgag cgggctggag cacctggccg tgaggcagtc ccctgcctgg 1800 agaattctgc ctgcaaagat agcagaggtc atggaggagc tgaaagccgt ggaggtcttc 1860 ctgaagagcg acagcctgtg tctgatggaa gggcggcgct tccgggcgca gcccaccctg 1920 ccctcggccc acctcctggc catgcacatc cagcagctgg agacaggcgg cttcaccatg 1980 accaacgggg cccacaggtg gagcaagctc aggaacatcg caaaggtggt gagccaggtg 2040 cacgcgttcc aggagaaccc ttacaccttc agccccgacc ccaagctcca gtcgtacctc 2100 aagcagagga ttgcccgctt cagcggtgcc gacatttcca cactcgccgc agatagcagg 2160 gccaacttcc accaggtctc cagcgagaag cactcacgga agattcagga caagctacgg 2220 aggatgaagg ctacattcca gtag 2244 <210> 3 < 211> 16 <212> PRT <213> Artificial Sequence <400> 3 Arg Ala Glu Gly Asn Pro Arg Gly Thr Asp Leu Glu Asn Pro Arg 5 10 15 Glu 16 <210> 4 <211> 10 <212> PRT <213 > Artificial Sequence <400> 4 Met Ala Arg Thr Ser Ser Arg Ala Pro Cys 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <400> 5 Asp Lys Leu Arg Arg Met Lys Ala Thr Phe Gln 5 10 <210> 6 <211> 48 <212> DNA <213> Artificial Sequence <400> 6 cgggccgagg gcaaccctcg cggcacagac ctggagaacc ccagggag 48 <210> 7 <211> 298 <212> DNA <213> HOMO SAPIENS <400> 5 gatccgaatc cgactgtggg gggcgggctg ggaggtggga gccgcgtct c 60 aggcccggcc gttatcaagg cccctccgcc cccgaaccct ggggagctgg 120 accaggaggt ggaggctcag gggaccccat ggggacaggc agagctggtc 180 tcctcccagc agacggagcc aggacgggca caagagtctt ggaggtttgc 240 gtgtttctgc tagaattaaa aagttaaatt taaaaatgaa aatgnaaa 298 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 cctccaagac tcttgtgccc gtcctggctc 30 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <400> 7 cgaaccactg aattccgcat tgcagag 27

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows that sequence-1 in FIG. 1 shows a high expression frequency in a recombinant obtained from a cerebral cortex cDNA library.
Represents an NA fragment, and sequences -2 to 3 are DNAs containing sequence-1.
The oligonucleotide used for the cloning of the A fragment is shown. Sequence-2 is a partial sequence of Sequence-1, and Sequence-3 is a partial sequence of lambda DR2 which is a vector of a cDNA library used for cloning. Sequence-4 represents the sequence of the oligopeptide used as the antigen.

FIG. 2 shows the nucleotide sequence of a DNA fragment containing the gene hucep-9 (1).

FIG. 3 shows the nucleotide sequence of a DNA fragment containing the gene hucep-9 (2).

FIG. 4 is a graph showing an increase in antibody titer.

FIG. 5 is a graph showing the results of antigen-specific IgG purification.

FIG. 6 shows the results of immunohistochemical staining.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/15 G01N 33/50 T 33/50 Z 33/53 D 33/53 C12N 15/00 ZNAA (72 ) Inventor Madoka Saito 3-24-1, Takada, Toshima-ku, Tokyo F-term in Taisho Pharmaceutical Co., Ltd. QA01 QA18 QA19 QQ02 QQ79 QQ96 QR32 QR40 QR48 QS15 QS33 4H045 AA10 AA11 AA20 AA30 BA10 CA45 DA75 DA86 EA50 FA71 FA74 GA26

Claims (13)

[Claims] 1. A protein of the following (a) or (b): (a) a protein consisting of the amino acid sequence of SEQ ID NO: 1; (b) one or several amino acids in the amino acid sequence of SEQ ID NO: 1 A protein comprising an amino acid sequence in which is deleted, substituted or added, and having guanine nucleotide exchange factor activity. 2. A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 1, and constituting a Lewy body. 3. A peptide comprising the amino acid sequence of any one of SEQ ID NOs: 3 to 5. A gene encoding the protein according to claim 1 or 2. 5. A gene consisting of the following (c) or (d); (c) a DNA consisting of the nucleotide sequence of SEQ ID NO: 2; (d) a DNA hybridizing with the DNA of SEQ ID NO: 2 under stringent conditions. DNA encoding soybean and encoding a protein having guanine nucleotide exchange factor activity. 6. A DNA which hybridizes with the DNA of SEQ ID NO: 2 under stringent conditions and encodes a protein constituting a Lewy body. 7. A DN comprising the nucleotide sequence of SEQ ID NO: 6.
A. An antibody against the protein according to claim 1 or 2. 9. An antibody against an oligopeptide having a part of the protein according to claim 1 or 2. 10. An antibody against the oligopeptide of any one of SEQ ID NOs: 3 to 5. [11] A test agent for Parkinson's disease, comprising the antibody according to [9] or [10]. 12. A method for screening a compound or a salt thereof that inhibits the aggregation of the protein according to claim 1 or 2. [13] A method for screening a compound or a salt thereof which promotes or inhibits the expression of the gene according to any one of [4] to [6].