JP2004041180A - New protein and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein and a DNA encoding the protein by analyzing the base sequence of a cDNA clone in a catalogized complete length cDNA library, specifying the physiological activity of the protein encoded by the cDNA when the sequence of the clone is new and using the physiological activity as a base for specifying the protein, etc. <P>SOLUTION: The protein is (a) a protein composed of a specific amino acid sequence or (b) a protein composed of an amino acid sequence obtained by the deletion, substitution and/or addition of one or more amino acids in the amino acid sequence of the protein (a) and having an RIM-binding activity. <P>COPYRIGHT: (C)2004,JPO

Description

【０１２９】
表１から明らかな通り、ｄｎａｆｏｒｍ４３０５９は脳での発現が高く、糖尿病の膵臓で発現が増加し、結腸癌で減少した。ｄｎａｆｏｒｍ５００３４は脳で発現が高く、糖尿病の膵臓で発現が増加し、結腸癌で減少した。これらの結果より、本発明のＤＮＡによってコードされるタンパク質は、上記のようなｍＲＮＡ発現の変動が見られる組織あるいはｍＲＮＡ発現量の多い組織に関わる疾患に関与している可能性があると考えられる。
【０１３０】
即ち、ｄｎａｆｏｒｍ４３０５９（配列番号１）によりコードされるタンパク質は、実施例４より、ＲＩＭ−ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎの１種であることが推測された。ＲＩＭ−ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎは神経伝達物質の分泌に関与していると考えられる。また、上記結果より、脳での発現が高く、糖尿病の膵臓で発現が増加し、結腸癌で減少していることも解った。以上のことから、本発明タンパク質の発現制御物質、機能賦活物質、あるいは機能阻害物質は、神経伝達の異常に関与する疾患、例えばアルツハイマー型痴呆、パーキンソン病、舞踏病、虚血性脳疾患、糖尿病性末梢神経障害などの治療薬として開発できる可能性がある。また、本発明の蛋白質は、糖尿病、癌等の治療薬として開発できる可能性がある。
【０１３１】
ｄｎａｆｏｒｍ５００３４（配列番号２）によりコードされるタンパク質は、実施例４より、ＲＩＭ−ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎの１種であることが推測された。ＲＩＭ−ｂｉｎｄｉｎｇ　ｐｒｏｔｅｉｎは神経伝達物質の分泌に関与していると考えられる。また、上記結果より、脳での発現が高く、糖尿病の膵臓で発現が増加し、結腸癌で減少していることも解った。以上のことから、本発明タンパク質の発現制御物質、機能賦活物質、あるいは機能阻害物質は、神経伝達の異常に関与する疾患、例えばアルツハイマー型痴呆、パーキンソン病、舞踏病、虚血性脳疾患、糖尿病性末梢神経障害などの治療薬として開発できる可能性がある。また、本発明の蛋白質は、糖尿病、癌等の治療薬として開発できる可能性がある。
【０１３２】
【発明の効果】
本発明のタンパク質は、ＲＩＭ結合活性を有することから、該タンパク質あるいはそれをコードするＤＮＡを用いて該活性を調節する物質をスクリーニングすることができ、該タンパク質が関連する疾患等に作用し得る医薬等の開発に有用である。また、本発明のＤＮＡおよび該ＤＮＡによってコードされるタンパク質は、糖尿病や癌などの治療や診断に応用できると考えられる。
本出願は、２００２年４月３０日付けの日本特許出願（特願２００２−１２８８０７）に基づくものであり、その内容はここに参照として取り込まれる。また、本明細書にて引用した文献の内容もここに参照として取り込まれる。
【０１３３】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel protein, a DNA encoding the protein, a full-length cDNA encoding the protein, a recombinant vector having the DNA, an oligonucleotide comprising a partial sequence of the DNA, and a transgenic cell into which the DNA has been introduced. And antibodies that specifically bind to the protein.
[0002]
[Prior art]
Obtaining cDNA and analyzing its base sequence are indispensable for analyzing the physiological activity of a protein expressed in a living body and developing a method for utilizing the protein based on the activity. Furthermore, creating a library in which full-length cDNAs corresponding to all gene types are cataloged is one of the important issues of the human genome project. The cataloged library means that there is no duplication in the cDNAs contained in the library, and refers to a library containing one type of each cDNA.
[0003]
The full-length cDNA cloning method is described in JP-A-9-248187 and JP-A-10-127291. This method comprises the steps of binding a molecule serving as a tag to a diol structure present at the 5 ′ cap site of mRNA, using the mRNA bound with the tag molecule as a template, and oligo-dT as a primer to reverse-transcribe the RNA-DNA complex. And separating the complex having a DNA corresponding to the full length of the mRNA using the function of the tag molecule.
[0004]
As an efficient reverse transcription method, a method for performing the transcription at a high temperature such that the template does not form a higher-order structure has been developed (Japanese Patent Laid-Open No. Hei 10-84661). Furthermore, a cloning vector has been developed which can uniformly clone a DNA fragment contained in a synthesized full-length cDNA library regardless of its chain length (Japanese Patent Application Laid-Open No. H11-9273).
[0005]
A full-length cDNA library produced by such a technique does not necessarily include all the elements that are different evenly as individual elements of the library, and is present only in clones with a high abundance ratio or, conversely, in very small amounts. There are clones. Since a clone present in only a trace amount is highly likely to be novel, a subtraction method and a normalization method for enriching such a clone have also been developed (Japanese Patent Laid-Open No. 2000-325080; Carninci, P. et al., Genomics, $ 37, $ 327-336 (1996)).
[0006]
The nucleotide sequence of each clone of the cataloged full-length cDNA library thus obtained can be identified by a known method. However, the physiological activity of the protein encoded by the cDNA remains unknown. It is.
[0007]
[Problems to be solved by the invention]
The present invention analyzes the nucleotide sequence of a cDNA clone contained in a cataloged full-length cDNA library, and among those having a novel sequence, identifies the biological activity of the protein encoded by the cDNA sequence and determines the biological activity. It is an object of the present invention to propose a method of using a protein based thereon and DNA encoding the same.
[0008]
[Means for Solving the Problems]
The present inventors analyzed the nucleotide sequence of the cDNA clone in the mouse full-length cDNA library and searched a database based on the homology of the sequence, and found that the sequence specific to a protein having RIM binding activity was The sequence was found and the proteins encoded by these cDNAs were identified as proteins having RIM binding activity. The expression level of the cDNA in each tissue was analyzed. The present invention has been achieved based on these findings.
[0009]
That is, according to the present invention, the following inventions (1) to (15) are provided.
(1) A protein of the following {(a)} or {(b)}.
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;
(B) 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: 3 or 4, and having RIM binding activity.
[0010]
(2) DNA encoding the protein of (1).
(3) A full-length cDNA encoding the protein of (1).
(4) DNA of any of the following (a), (b) or (c):
(A) DNA having the nucleotide sequence of SEQ ID NO: 1 or 2.
(B) a DNA having a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence of SEQ ID NO: 1 or 2, and which encodes a protein having RIM binding activity.
(C) a DNA having a base sequence capable of hybridizing under stringent conditions with a DNA having the base sequence of SEQ ID NO: 1 or 2 or a sequence complementary thereto and encoding a protein having RIM binding activity .
[0011]
(5) A recombinant vector containing the DNA according to any one of (2) to (4).
(6) A gene-transfected cell into which the DNA according to any one of (2) to (4) or the recombinant vector according to (5) has been introduced, or an individual comprising the cell.
(7) The protein according to (1), which is produced by the cell according to (6).
[0012]
(8) A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA according to any of (2) to (4), and an antisense having a sequence complementary to the sense oligonucleotide. An oligonucleotide selected from the group consisting of a sense oligonucleotide and an oligonucleotide derivative of the sense or antisense oligonucleotide.
[0013]
(9) An antibody or a partial fragment thereof that specifically binds to the protein of (1) or (7).
(10) The antibody according to (9), wherein the antibody is a monoclonal antibody.
(11) The antibody according to (10), wherein the monoclonal antibody has an action of neutralizing the RIM binding activity of the protein according to (1) or (7).
[0014]
(12) の A method for controlling an activity of a protein, comprising bringing the protein according to (1) or (7) into contact with a test substance and measuring a change in the activity of the protein caused by the test substance. Screening method.
(13) Screening for a substance regulating the expression of DNA, which comprises bringing the test substance into contact with the gene-introduced cell according to (6) and detecting a change in the expression level of the DNA introduced into the cell. Method.
(14) At least one or more amino acid sequence information selected from the amino acid sequence of the protein described in (1) and / or at least one selected from the nucleotide sequence of the DNA described in any of (2) to (4). A computer-readable recording medium storing one or more base sequence information.
(15) A carrier to which the protein according to (1) and / or the DNA according to any of (2) to (4) are bound.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in more detail.
(1) Acquisition of full-length cDNA and analysis of nucleotide sequence
The DNA of the present invention is a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4, or one or several of the same in the amino acid sequence (the number is not particularly limited, for example, 20 or less, Preferably 15 or less, more preferably 10 or less, and still more preferably 5 or less) amino acid residue substitution, deletion, insertion, addition, or inversion, and RIM binding Any protein can be used as long as it can encode a protein having activity. Specifically, it may be only the translation region encoding the amino acid sequence or may include the full length of the cDNA.
[0016]
Specifically, examples of the DNA containing the full-length cDNA include a DNA having the nucleotide sequence of SEQ ID NO: 1 or 2. Examples of the translation region include those having sequences represented by base numbers 297 to 2507 of SEQ ID NO: 1 and base numbers 724 to 3939 of SEQ ID NO: 2. Further, the DNA of the present invention includes not only the full length of the above-mentioned cDNA but also those containing the above-mentioned translation region and a portion adjacent to the 3 'and / or 5' end thereof, which is the minimum necessary for the expression of the translation region. .
[0017]
The DNA of the present invention may be obtained by any method as long as it can be obtained, and specifically, can be obtained, for example, by the following method. First, mRNA is prepared from a suitable animal, preferably a mammalian tissue or the like by a method known per se and generally used. Next, cDNA is synthesized using this mRNA as a template. At this time, in order to synthesize a full-length cDNA, a molecule serving as a tag is chemically bonded to a diol structure specific to a 5 ′ cap (7MeGpppN) site. Using a method of separating the full-length cDNA using the function of the tag molecule after reverse transcription using oligo dT as a primer with as a template (JP-A-9-248187; JP-A-10-127291) Is preferred. In addition, in the case of reverse transcription, in order to prevent the template from forming a higher-order structure and lowering the efficiency of reverse transcription, in the presence of trehalose or the like, use a thermostable reverse transcriptase at a high temperature. It is preferable to use a method of performing reverse transfer (Japanese Patent Laid-Open No. 10-84661). Here, high temperature means 40-80 degreeC.
[0018]
The thus obtained cDNA is cloned by inserting it into an appropriate cloning vector. The vector used herein has a recombination recognition sequence at both ends of a cloning site capable of uniformly cloning DNAs of various chain lengths, and is a linear vector inserted into a host by a method other than infection. (JP-A-11-9273) is preferably used. In the cDNA library thus obtained, not all clones exist uniformly (hereinafter, this may be referred to as "cataloged"), but only a very small amount exists in this library. A clone that does not have a high probability of being new. Therefore, it is preferable to use a subtraction method or a normalization method for enriching such clones (Japanese Patent Application Laid-Open No. 2000-325080; Carinci, P. et al., Genomics, # 37, # 327-336 (1996)).
[0019]
The nucleotide sequence of the cataloged cDNA library is analyzed by a commonly used method known per se. In the case of the DNA of the present invention, in the case of the full-length cDNA, the base sequence obtained from the sequence based on the terminal 100 is obtained by combining BLAST (http://www.ncbi.nlm.nih.gov/BLAST/; National Center Center of Biotechnology Information). ), NCBI databases such as Genbank, EMBL, and DDBJ were searched, and even the sequence showing the highest homology was found to have a similarity of 30% or less as a new sequence for the following analysis.
[0020]
Examples of the DNA having such a full-length cDNA base sequence include a DNA having the base sequence of SEQ ID NO: 1 or 2 as described above. Examples of the translation region include those having sequences represented by base numbers 297 to 2507 of SEQ ID NO: 1 and base numbers 724 to 3939 of SEQ ID NO: 2.
[0021]
The novel base sequence thus obtained is subjected to homology search by BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990)). HMMPF (HMMPFAM) (sequence analysis method using hidden Markov model; {Eddy, {S.} R., {Bioinformatics} 14, {755-673} (1998))} {protein feature search by HMMPFAM} (profile search: //Pfam.wustl.edu) or the like, the function of the protein encoded by the nucleotide sequence can be estimated.
[0022]
In the homology search by BLAST, the function of the clone to be analyzed can be estimated from various annotation information associated with the hit sequence whose homology is sufficiently significant as a result of the search. Here, a sufficiently significant hit sequence means that the degree of coincidence between the functional domain portion of the registered amino acid sequence and the corresponding portion of the amino acid sequence encoded by the DNA of the present invention is 10 as an e-value.^-4Show the following or 30% or more.
[0023]
For example, if most of the top-ranked functional domain sequences have been confirmed to have RIM binding activity, the clone to be analyzed that is similar in sequence to that will also have the same function, that is, RIM binding activity. The prediction holds.
Here, in the present invention, the protein having RIM binding activity means a protein capable of binding to RIM (Rab3-interacting molecule) which is a target protein of Rab3A, that is, a RIM-binding protein (RIM-binding protein). I do.
[0024]
In the HMMPFAM, an analysis is performed by a method of examining whether a sequence to be analyzed has a feature of a sequence included in an entry in a database in which a protein profile called Pfam is accumulated. Profiles are extracted from a series of proteins with the same characteristics, and even if the structure cannot be determined by comparing one-to-one sequences over the entire length, if there is a characteristic region in the sequence, it can be found and its function can be predicted. . A specific example of the protein function prediction performed in this manner will be described below.
[0025]
The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 was identified by BLAST search with KIAA0318 gene, partial cds (Homo sapiens) and e-value: 0, 725 amino acid residues at 75% identity, and RIM. -Binding protein 2 (chicken) and e-value: 72% identity over 0,722 amino acid residues, and furthermore, peripheral benzodiazepine receptor accepting protein (Homo sapiens): e-v10 and e-v10.⁻¹¹⁸, 376 amino acid residues with a 37% match.
From these results, it is inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 is a kind of RIM-binding @ protein.
Since RIM-binding protein is considered to be involved in the secretion of neurotransmitters, the protein of the present invention may be used as an expression regulator, a function activator, or a function inhibitor for diseases associated with abnormal neurotransmission, such as Alzheimer's disease. It may be a therapeutic drug for type dementia, Parkinson's disease, chorea, ischemic brain disease, diabetic peripheral neuropathy and the like.
[0026]
The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 2 was compared with KIAA0318 gene, partial cds (Homo sapiens) and e-value: 0, 1077 amino acid residues by BLAST by 81%, and RIM- binding-protein-2 (chicken) and e-value: 75% identity over 0,813 amino acid residues, and further RIM binding protein 1A (Rbp1A) mRNA, partial cds (Rattus) and e-value: 5 × 10.⁻¹¹¹, 843 amino acid residues with a 35% match.
From these results, it is presumed that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 2 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 is one kind of RIM-binding @ protein.
Since RIM-binding protein is considered to be involved in the secretion of neurotransmitters, expression control substances, function activators or function inhibitors of the protein of the present invention may be used in diseases involving abnormal neurotransmission, such as Alzheimer's type. It may be a therapeutic drug for dementia, Parkinson's disease, chorea, ischemic brain disease, diabetic peripheral neuropathy and the like.
[0027]
The DNA of the present invention may be obtained in a state where a base sequence is deleted or inserted in the translated sequence. As a result of performing the homology search or the protein feature search as described above, the base sequence of the DNA is determined. If a deletion or insertion in the DNA is estimated, it is possible to obtain a full-length cDNA having no base deletion or insertion by using a method commonly used in the art such as library screening or PCR cloning. it can. The protein of the present invention is expressed using the full-length cDNA thus obtained, and can be used for functional analysis.
[0028]
The DNA of the present invention thus obtained, whose nucleotide sequence is determined, and whose function is presumed, includes only the nucleotide sequence of SEQ ID NO: 1 or 2 or the nucleotide having the nucleotide sequence shown above as its translation region. In these base sequences, one or several (the number is not particularly limited, but is, for example, 60 or less, preferably 30 or less, more preferably 20 or less, and still more preferably 10 or less) , Particularly preferably 5 or less), a DNA encoding a protein having a base sequence with deletion, substitution, and / or addition of a base and having RIM binding activity; A DNA that hybridizes with a complementary sequence under stringent conditions and encodes a protein having RIM binding activity is also included. As described above, these DNAs have an amino acid sequence in which one or several amino acid sequences are deleted, substituted and / or added in the amino acid sequence of the protein of SEQ ID NO: 3 or 4, and have RIM binding activity. Includes those that encode proteins.
[0029]
Here, DNA that hybridizes under stringent conditions refers to the nucleotide sequence of SEQ ID NO: 1 or 2 or a complementary sequence thereof to 80% or more, preferably 90% or more, and more preferably 95% or more by BLAST analysis. DNA containing a base sequence having homology to Further, the hybridization under stringent conditions means that the reaction is carried out in a normal hybridization buffer at a temperature of 40 to 70 ° C, preferably 60 to 65 ° C, and a salt concentration of 15 mM to 300 mM, preferably The washing can be performed according to a method of washing in a washing solution of 15 mM to 60 mM or the like.
[0030]
Further, the DNA of the present invention may be obtained by the above-described method or may be synthesized. The DNA base sequence can be easily replaced with a commercially available kit such as a site-directed mutagenesis kit (Takara Shuzo) or a quick change site-directed mutagenesis kit (Stratagene).
[0031]
The nucleotide sequence set forth in SEQ ID NO: 1 or 2 is derived from a mouse. A human cDNA library is prepared according to the above-described method for preparing a cDNA library, and Obtaining DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence represented by SEQ ID NO: 1 or 2 by performing hybridization using a DNA fragment having the nucleotide sequence represented by 1 or 2 as a probe You can also. The DNA that hybridizes with the DNA of SEQ ID NO: 1 or 2 or a complementary sequence thereof under stringent conditions includes such a human homologous DNA and a human orthologous DNA described below.
[0032]
Further, it is also possible to predict the base sequence of the human homolog DNA by using informatics, and to obtain the human homolog DNA from the above-mentioned human cDNA library or the like based on the base sequence.
[0033]
In general, as a method for predicting a base sequence encoding a homolog protein of a target protein using informatics, for example, (i) using a base sequence of a target cDNA as a query, A method of performing a homology search on a database (including a cDNA database predicted by informatics) using BLAST or the like; and (ii) BLAST against a human or other EST database using the base sequence of the target cDNA as a query. A method of performing homology search using such a method and linking the sequence of the hit EST with reference to the base sequence of the target cDNA, and (iii) using the base sequence of the target cDNA as a query Homology search for genome database using BLAST etc. In addition, the position on the genome where the gene of the cDNA of interest exists is specified, and Genscan (http: //@genes.@mit.edu/GENSCAN.html) or Sim4 (Genome @ Res., # 8: 977-74 (1998)) and the like, and a method of predicting the nucleotide sequence of a gene portion in the genome.
[0034]
When predicting the nucleotide sequence of the human homolog DNA of mouse-derived cDNA, any of the above methods can be used. However, the cDNA having the nucleotide sequence of SEQ ID NO: 1 or 2 of the present invention is novel, In the method (2), it is considered that the nucleotide sequence of the human homologous DNA cannot be obtained. Therefore, it is preferable to use the method described in (ii) or (iii).
[0035]
Based on the predicted nucleotide sequence of the human homolog DNA, a DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence of SEQ ID NO: 1 or 2 can be obtained from the above human cDNA library. . As a specific acquisition method, for example, PCR is performed using the above human cDNA library as a template, using a primer having a nucleotide sequence complementary to the nucleotide sequence at the 5 ′ end and 3 ′ end of the predicted human homolog DNA. And a method of performing hybridization with the above human cDNA library using a partial sequence of the predicted human homolog DNA as a probe.
[0036]
Generally, a similar gene having a nucleotide sequence having a high homology with the nucleotide sequence of the target gene is referred to as a “homolog”, and the above-described method also aims to obtain a human homolog. It is important to confirm that not only the similarity but also that the gene obtained as a homolog is a family member of the target gene. Genes acquired as "homologs" between two species of organisms are likely to be "orthologs", the same gene evolved from a common ancestral gene, and differ from each other caused by duplication from a common ancestral gene It may be a "paralog" that is a gene.
[0037]
That is, in order for the human-derived DNA obtained as a homologue to have the same function as the protein of the present invention, the function of the protein encoded by the human-derived DNA must be In order to verify the function of the protein of the present invention as a mouse, it is preferable to confirm that the human homolog is an ortholog of a closely related species of the mouse gene of the present invention.
[0038]
For example, the following method is used as a method for confirming the ortholog. (I) First, homology is analyzed between the nucleotide sequence of the cDNA of the present invention and the nucleotide sequence of the obtained human homolog DNA. Next, using the base sequence of the cDNA of the present invention as a query, a homology search was performed on human base sequences contained in international base sequence databases such as DDBJ, EMBL, GenBank, and patent databases, and the base sequence of the obtained human homolog DNA was obtained. And that the base sequence of the query is higher than the base sequence obtained from the database. Further, (ii) homology is analyzed between the nucleotide sequence of the obtained human homolog DNA and the corresponding nucleotide sequence of the cDNA of the present invention. Next, using the base sequence of the obtained human homologous DNA as a query, a homology search was performed for the mouse base sequence contained in the international base sequence database such as DDBJ, EMBL, GenBank, and the patent database, and the base sequence of the cDNA of the present invention was obtained. And that the base sequence of the query is higher than the base sequence obtained from the database and the base sequence of the query. By confirming the above (i) and (ii), the obtained human homolog can be identified as a human ortholog corresponding to the cDNA of the present invention. The homology analysis described in (i) and (ii) above may be performed by comparing amino acid sequences, or by drawing a molecular evolutionary phylogenetic tree and examining it. In addition, it is preferable that the degree of coincidence by the homology analysis described in the above (i) and (ii) be analyzed as the degree of coincidence over the entire length of the query.
[0039]
By performing a homology search by BLAST or a protein feature search by HMMPFAM on the nucleotide sequence of the human homologous DNA or orthologous DNA thus obtained, the function of the protein encoded by the nucleotide sequence can be estimated.
Furthermore, the protein of the present invention is expressed using the obtained full-length cDNA of human homolog DNA or human ortholog DNA, and can be used for confirmation of activity, functional analysis, and the like.
[0040]
(2) Protein encoded by the novel cDNA
The translation region of the protein encoded by the DNA of the present invention is, for example, a base sequence of the DNA which is converted into amino acids by three types of reading frames, and the range encoding the longest polypeptide is defined as the translation region of the present invention. The amino acid sequence can be determined. Examples of such an amino acid sequence include those described in SEQ ID NO: 3 or 4. Further, the protein of the present invention is not limited to the above amino acid sequence, but comprises an amino acid sequence in which one or several amino acids have been substituted, deleted and / or added, and has an RIM binding activity. And those having the following.
[0041]
As a method for obtaining the protein of the present invention, the method of transcription / translation of the DNA of the present invention described in the above (1) by an appropriate method is preferably used. Specifically, a recombinant vector inserted into a suitable expression vector or a suitable vector together with a suitable promoter is prepared, and a suitable host microorganism is transformed with the recombinant vector or introduced into a suitable cultured cell. And then can be obtained by purification.
[0042]
When the protein thus obtained is obtained in a free form, it can be converted to a salt by a known method or a method analogous thereto, and conversely, when the protein is obtained in a salt form, it can be converted to a free form or another salt. can do. Such salts of the protein of the present invention are also included in the protein of the present invention. Further, a protein produced by the transformant may be modified before or after purification by applying an appropriate protein modifying enzyme to modify the protein arbitrarily or partially removing the polypeptide. Can be. These modified proteins are also included in the scope of the present invention as long as they have RIM binding activity.
[0043]
When producing the protein of the present invention, the vector used for the production of the recombinant vector containing the DNA of the present invention is not particularly limited as long as the DNA is expressed in the transformant. Any of vectors may be used. Of these, usually, a commercially available protein expression vector into which an expression control region DNA such as a promoter suitable for a host into which the DNA is introduced has already been inserted is used. Specific examples of such a protein expression vector include pET3 and pET11 (manufactured by Stratagene) and pGEX (manufactured by Amersham Pharmacia Biotech) when the host is Escherichia coli, and pESP when the host is yeast. -I expression vector (manufactured by Stratagene) and the like, and in the case of insect cells, BacPAK6 (manufactured by Clontech). When the host is an animal cell, examples include ZAP @ Express (manufactured by Stratagene), pSVK3 (manufactured by Amersham Pharmacia Biotech), and the like.
[0044]
When using a vector into which the expression control region has not been inserted, it is necessary to insert at least a promoter as the expression control region. Here, as the promoter, a promoter contained in a host microorganism or a cultured cell can be used. However, the promoter is not limited thereto. Specifically, for example, when the host is Escherichia coli, T3, T7, tac Lac promoter and the like, and in the case of yeast, for example, nmt1 promoter, Gal1 promoter and the like can be used. When the host is an animal cell, SV40 promoter, CMV promoter and the like are preferably used.
[0045]
When a host capable of functioning with a mammalian-derived promoter is used, a promoter specific to the gene of the present invention can also be used. Insertion of the DNA of the present invention into these vectors may be performed by linking the DNA or a DNA fragment containing the DNA to the amino acid sequence of the protein encoded by the gene DNA downstream of the promoter in the vector.
[0046]
The recombinant vector thus prepared can be used to transform a host described below by a method known per se to prepare a DNA transductant. As a method for introducing the vector into a host, specifically, a heat shock method (J. @Mol. Biol., 53, 154 (1970)), a calcium phosphate method (Science, 221, 551, @ (1983)), DEAE Dextran method (Science, 215, 166, (1982)), in vitro packaging method (Proc. Natl. Acad. @ Sci. USA, 72, 581, (1975)), virus vector method (Cell, 37, 1053, (1984)) )), And the electric pulse method (Chu. Et al., Nuc. Acids Res., 15, 1331 (1987)).
[0047]
The host for preparing the DNA transfectant is not particularly limited as long as the DNA of the present invention is expressed in the body. For example, Escherichia coli, yeast, baculovirus (arthropod polyhedrosis virus) -insect cells, or Animal cells and the like can be mentioned. Specifically, BL21 and XL-2Blue (manufactured by Stratagene) and the like for Escherichia coli, SP-Q01 (manufactured by Stratagene) and the like for yeast, and AcNPV (J. Biol. Chem., 263, 7406, and the like) for baculovirus. 1988)) and its host Sf-9 cells (J. Biol. Chem., 263, 7406, (1988)). Examples of animal cells include mouse fibroblast C127 (J. Viol., 26, 291, (1978)) and Chinese hamster ovary cell CHO cells (Proc. Natl. Acad. Sci. USA, 77, 4216, (1980)). Among them, COS-7 cells derived from African green monkey kidney (ATCC @ CRL1651: American Type Culture Collection preserved cells), HEK293 cells derived from human fetal kidney (ATCC @ CRL1573) or Human cervical cancer HeLa cells (ATCC @ CCL-2) are used.
[0048]
In addition to the expression method using the protein expression vector as described above, a homologous recombination technique (AA Vertes @ et @ al., Biosci.) In which a DNA fragment of the present invention linked to a promoter is directly inserted into the chromosome of a host microorganism. Biotechnol. Biochem., 57, 2036, (1993)) or a transposon or an insertion sequence (AA Vertes et al., Molecular Microbiol., 11, 739, 1994). It can also be made.
[0049]
The obtained culture is obtained by collecting cells or cells by a method such as centrifugation, suspending the cells or the like in a suitable buffer, and sonicating, lysozyme, and / or freezing and thawing. After the disruption, a crude protein solution is obtained by centrifugation, filtration, or the like, and further purified by a combination of appropriate purification methods. Thus, the protein of the present invention is obtained. In addition to the above-described expression method using the protein expression recombinant vector, protein expression is induced by subjecting the DNA of the present invention obtained in (1) to a cell-free transcription / translation system to obtain the protein of the present invention. be able to. The cell-free transcription / translation system used in the present invention is a system containing all the elements necessary for transcription from DNA to mRNA and translation from mRNA to protein. Refers to any system by which the protein being synthesized is synthesized. Specific examples of the cell-free transcription / translation system include a transcription / translation system prepared based on an eukaryotic cell and a bacterial cell, or an extract from a part thereof. A transcription / translation system prepared based on an extract from Erythrocytes, wheat germ and Escherichia coli (Escherichia coli S30 extract) may be mentioned.
[0050]
Separation and purification of the protein of the present invention from the obtained transcription / translation product of the cell-free transcription / translation system can be carried out by a commonly used method known per se. Specifically, for example, a DNA region encoding an epitope peptide, a polyhistidine peptide, glutathione-S-transferase (GST), a maltose binding protein, or the like is introduced into the DNA to be transcribed and translated, and expressed as described above. The protein can be purified by utilizing the affinity of the protein with a substance having affinity.
[0051]
The expression of the target protein is separated by SDS-polyacrylamide gel electrophoresis or the like, and stained with Coomassie Brilliant Blue (manufactured by Sigma) or detected by an antibody that specifically binds to the protein of the present invention described later. It can be confirmed by the method of performing. In general, it is known that an expressed protein is cleaved (processed) by a proteolytic enzyme present in a living body. The protein of the present invention is naturally included in the protein of the present invention as long as it has a RIM binding activity, even if it is a partial fragment of the truncated amino acid sequence.
[0052]
By analyzing the interaction between the thus obtained protein and other proteins and DNA, it is possible to know the multifaceted functions in the living body. As a method for analyzing the interaction, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosomal method Display method and the like can be mentioned.
[0053]
(3) Preparation of oligonucleotide
Using the DNA of the present invention or a fragment thereof obtained by the method described in (1) above, an antisense oligonucleotide having a partial sequence of the DNA of the present invention, a sense -An oligonucleotide such as an oligonucleotide can be prepared.
[0054]
Examples of the oligonucleotide include a DNA having the same sequence as 5 to 100 consecutive bases in the base sequence of the DNA or a DNA having a sequence complementary to the DNA. Specific examples include a DNA having the same sequence as 5 to 100 consecutive nucleotides in the base sequence represented by SEQ ID NO: 1 or a DNA having a sequence complementary to the DNA. When used as a sense primer and an antisense primer, the above-mentioned oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not extremely change are preferable. The length of the sequence is generally 5 to 100 bases, preferably 10 to 60 bases, and more preferably 15 to 50 bases.
[0055]
In addition, derivatives of these oligonucleotides can also be used as the oligonucleotide of the present invention. Examples of the oligonucleotide derivative include an oligonucleotide derivative in which a phosphoric diester bond in an oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in which a phosphoric diester bond in an oligonucleotide is converted to an N3′-P5 ′ phosphoramidate bond. Nucleotide derivative, oligonucleotide derivative in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, oligonucleotide derivative in which uracil in oligonucleotide is substituted with C-5 propynyluracil, uracil in oligonucleotide is Oligonucleotide derivatives substituted with C-5 thiazole uracil, oligonucleotide derivatives substituted with cytosine in the oligonucleotide with C-5 propynylcytosine, oligonucleotides Is an oligonucleotide derivative in which cytosine is substituted by phenoxazine-modified cytosine, an oligonucleotide derivative in which ribose in the oligonucleotide is substituted by 2′-O-propylribose, or ribose in the oligonucleotide is 2 Oligonucleotide derivatives substituted with '-methoxyethoxyribose can be mentioned.
[0056]
In addition, the oligonucleotide of the present invention can be prepared as a double-stranded RNA, introduced into a recipient, and used in an RNA interference method for inhibiting the expression of a target gene. As the RNA interference method, for example, a method described in (Elbashir, S., et al., Nature, 411, 494-498 (2001)) can be used. The double-stranded RNA does not necessarily have to be all RNA, and for example, those described in WO 02/10374 can be used.
[0057]
Here, the target gene may be any DNA as long as it is the DNA of the present invention. A double-stranded RNA consisting of a sequence substantially identical to at least a part of the base sequence of these DNAs (hereinafter sometimes referred to as “double-stranded polynucleotide”) is defined as a part of the base sequence of the target gene. And a sequence substantially the same as a sequence of 15 bp or more, which may be any part. Here, “substantially the same” means that it has 80% or more homology with the sequence of the target gene. The nucleotide length of the nucleotide may be any length from 15 bp to the entire length of the open reading frame (ORF) of the target gene, but a length of about 15 to 500 bp is preferably used. However, it is known that mammalian-derived cells have a signal transduction system activated in response to a long double-stranded RNA of 30 bp or more. This is called an interferon reaction (Mareus, P. I., et al., Interferon, 5, 115-180 (1983)), and when the double-stranded RNA enters a cell, PKR (dsRNA-responsive) is obtained. Initiation of translation of many genes is non-specifically inhibited via protein kinase: Bass, BL, Nature, 411, ２８428-429 (2001)), and at the same time, 2 ′, 5 ′ oligoadenylate synthetase (Bass, B.L., Nature, 411, ２８428-429 (2001)), RNaseL is activated, and non-specific degradation of intracellular RNA is caused. These non-specific reactions mask the specific response of the target gene. Therefore, when a mammal, or a cell or tissue derived from the animal is used as a recipient, a double-stranded polynucleotide of 15 to 30 bp, preferably 19 to 24 bp, and most preferably 21 bp is preferably used. The double-stranded polynucleotide does not need to be entirely double-stranded, and includes those in which the 5 'or 3' end is partially protruded, but those in which the 3 'end is protruded by two bases are preferably used.
[0058]
The double-stranded polynucleotide means a double-stranded polynucleotide having complementarity, but may be a self-annealed single-stranded polynucleotide having self-complementarity. The single-stranded polynucleotide having self-complementarity includes, for example, one having an inverted repeat sequence.
[0059]
The method for preparing the double-stranded polynucleotide is not particularly limited, but a known chemical synthesis method is preferably used. In chemical synthesis, a single-stranded polynucleotide having complementarity can be separately synthesized, and can be converted into a double-stranded strand by associating them by an appropriate method. Specific examples of the method of association include a method in which the synthesized single-stranded polynucleotide is mixed, heated to a temperature at which the double-strand is dissociated, and then gradually cooled. The associated double-stranded polynucleotide is confirmed using an agarose gel or the like, and the remaining single-stranded polynucleotide is removed by, for example, decomposing it with an appropriate enzyme.
[0060]
The transfectant into which the double-stranded polynucleotide thus prepared is introduced may be any as long as the target gene can be transcribed into RNA or translated into protein in the cell. Specific examples include those belonging to plant, animal, protozoan, virus, bacterial, or fungal species. The plant may be a monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate. Preferred microorganisms are those used in agriculture or industry, and those that are pathogenic for plants or animals. Fungi include organisms in both mold and yeast forms. Examples of vertebrates include mammals, including fish, cows, goats, pigs, sheep, hamsters, mice, rats and humans, and invertebrates include nematodes and other reptiles, Drosophila melanogaster ( Drosophila), and other insects. Preferably, the cells are vertebrate cells.
[0061]
The transductant means a cell, tissue, or individual. Here, the cell may be from germline or somatic, totipotent, or pluripotent, split or undivided, parenchymal tissue or epithelium, immortalized or transformed, and the like. The cells may be gametes or embryos, in the case of embryos, single-cell or constitutive cells, or cells from multi-cell embryos, including fetal tissue. Furthermore, they may be undifferentiated cells, such as stem cells, or differentiated cells, such as from cells of an organ or tissue, including fetal tissue, or any other cells present in an organism. Differentiating cell types include adipocytes, fibroblasts, muscle cells, cardiomyocytes, endothelial cells, nerve cells, glial, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, eosinophils, Includes basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes and cells of the endocrine or exocrine glands.
[0062]
As a method for introducing a double-stranded polynucleotide into a transfectant, when the transfectant is a cell or tissue, calcium phosphate method, electroporation method, lipofection method, virus infection, double-stranded polynucleotide solution Immersion, transformation, or the like. Examples of a method for introducing the gene into an embryo include microinjection, electroporation, and virus infection. When the recipient is a plant, a method of injecting or perfusing the plant into a body cavity or stromal cells, or spraying is used. In the case of an individual animal, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ocular and intraperitoneal administration. A method, an electroporation method, a virus infection, or the like is used. For methods for oral introduction, the double-stranded polynucleotide can be mixed directly with the food of the organism. Furthermore, when introduced into an individual, it can be administered, for example, by administration as an implanted long-term release preparation or the like, or by ingesting an introduced body into which a double-stranded polynucleotide has been introduced.
[0063]
The amount of the double-stranded polynucleotide to be introduced can be appropriately selected depending on the introduced substance and the target gene, but it is preferable to introduce an amount sufficient to introduce at least one copy per cell. Specifically, for example, when the transfectant is a human cultured cell and the double-stranded polynucleotide is introduced by the calcium phosphate method, 0.1 to 1000 nM is preferable.
By suppressing the expression of the gene of the present invention in the transfectant by RNA interference, the function of the protein encoded by the gene of the present invention can be confirmed, or a new function can be analyzed.
[0064]
(4) Antibodies that specifically bind to the protein of the present invention
As a method for preparing an antibody that specifically binds to the protein of the present invention, a commonly used known method can be used. For a polypeptide used as an antigen, an epitope (antigen determination An appropriate sequence can be selected and used as the group. As a method for selecting an epitope, for example, commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co., Ltd.) can be used.
[0065]
As the polypeptide used as the above antigen, a synthetic peptide synthesized according to a known method or the protein of the present invention itself can be used. The polypeptide serving as an antigen may be prepared in an appropriate solution or the like according to a known method and immunized to a mammal, for example, a rabbit, a mouse, a rat, or the like. It is preferable to use an antigen peptide as a conjugate with a suitable carrier protein or to add an adjuvant or the like for immunization.
[0066]
The route of administration of the antigen upon immunization is not particularly limited, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular may be used. Specifically, for example, a method of inoculating BALB / c mice several times every several days to several weeks with an antigen polypeptide is used. The amount of the antigen to be taken is preferably about 0.3 to 0.5 mg / time when the antigen is a polypeptide, but is appropriately adjusted depending on the type of the polypeptide and the animal species to be immunized.
[0067]
After immunization, blood is appropriately collected as a test, and an increase in the antibody titer is confirmed by a method such as enzyme-linked immunosorbent assay (hereinafter sometimes referred to as “ELISA”) or Western blotting. Blood is collected from animals with increased titers. A polyclonal antibody can be obtained by subjecting this to an appropriate treatment used for antibody preparation. Specifically, for example, there is a method of obtaining a purified antibody obtained by purifying an antibody component from serum according to a known method. For purification of the antibody component, methods such as centrifugation, ion exchange chromatography, and affinity chromatography can be used.
[0068]
A monoclonal antibody can also be prepared by using a hybridoma fused with spleen cells and myeloma cells of the animal according to a known method (Milstein, et al., Nature, 256, 495 (1975)). . The monoclonal antibody can be obtained, for example, by the following method.
[0069]
First, antibody-producing cells are obtained from an animal whose antibody titer has increased due to immunization with the above-described antigen. The antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any of the individuals, but is preferably obtained from the spleen, lymph nodes, peripheral blood and the like. As a myeloma to be fused with these cells, generally, a cell line obtained from a mouse, for example, a P3X63-Ag8.653 (ATCC: CRL) which is an 8-azaguanine-resistant mouse (derived from BALB / c) myeloma cell line is used. -1580), P3-NS1 / 1Ag4.1 (RIKEN cell bank: RCB0095) and the like are preferably used. For cell fusion, antibody-producing cells and myeloma cells are mixed at an appropriate ratio, and 50% polyethylene is added to an appropriate cell fusion medium such as RPMI1640, Iskov's modified Dulbecco's medium (IMDM), or Dulbecco's modified Eagle's medium (DMEM). It can be carried out by using a solution in which glycol (PEG) is dissolved. It can also be carried out by the electrofusion method (U. Zimmer-mann. Et al., Naturewissenschaften, 68, 577 (1981)).
[0070]
Hybridomas were prepared using a myeloma cell line that was resistant to 8-azaguanine, using 5% CO2 in a normal medium (HAT medium) containing an appropriate amount of hypoxanthine / aminopterin / thymidine (HAT) solution.₂At 37 ° C. for an appropriate time. This selection method can be appropriately selected and used depending on the myeloma cell line to be used. The antibody titer of the antibody produced by the selected hybridoma is analyzed by the above-described method, the hybridoma producing the antibody having a high antibody titer is separated by a limiting dilution method or the like, and the separated fused cells are cultured in an appropriate medium. The monoclonal supernatant can be obtained by purifying the resulting culture supernatant by an appropriate method such as ammonium sulfate fractionation and affinity chromatography. For purification, a commercially available monoclonal antibody purification kit can also be used. Furthermore, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or a nude mouse.
[0071]
In addition, when a human-derived protein is obtained as the protein of the present invention, the above-described method is applied to a Severe-combined-immune-deficiency (SCID) mouse transplanted with human peripheral blood lymphocytes using the polypeptide or a partial peptide thereof as an antigen. A humanized antibody can also be prepared by immunizing in the same manner as described above and preparing a hybridoma between the antibody-producing cells of the immunized animal and human myeloma cells (Mosier, D. E., et al. Nature, 335, 256-259} (1988); Duchosal, M.A., et al., Nature, 355, 258-262 (1992)).
[0072]
Further, RNA is extracted from the obtained hybridoma producing the human antibody, a gene encoding the target human antibody is cloned, this gene is inserted into an appropriate vector, and this is introduced into an appropriate host. By expression, human antibodies can be produced in larger quantities. Here, an antibody with low binding to an antigen can be obtained as an antibody with even higher binding by using an evolutionary engineering technique known per se. A partial fragment such as a monovalent antibody can be prepared by cleaving the Fab and Fc portions using, for example, papain or the like, and collecting the Fab portion using an affinity column or the like.
[0073]
The antibody that specifically binds to the protein of the present invention thus obtained can also be used as a neutralizing antibody that specifically binds to the protein of the present invention and thereby inhibits the RIM binding activity of the protein. There is no particular limitation on the method of selecting a substance that inhibits the activity of the protein. For example, it is possible to contact an antibody with the DNA transfectant prepared in (2) above and determine whether the function of the target protein in the transfectant is inhibited. And a method of analyzing the above.
[0074]
Such a neutralizing antibody may be used alone when the clinical application is performed, or may be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0075]
(5) Confirmation of activity and analysis of function of the protein of the present invention
The protein of the present invention is prepared as a recombinant protein as described in the above (2), and by analyzing this, it can be confirmed that it has the activity estimated in the above (1). Furthermore, analysis can also be performed by combination with the antibody or the like prepared as described in (4) above.
[0076]
Here, the Rab family protein, one of the low molecular weight GTP-binding proteins, is an important factor that controls intracellular vesicle trafficking in eukaryotic cells. In particular, Rab3A is abundantly expressed in the brain and concentrated in synaptic vesicles, and is involved in regulatory secretion including neurotransmitter release. RIM (Rab3-interacting @molecule), which is a target protein of Rab3A, is a macromolecule consisting of several hundreds of amino acids, and has zinc @ finger @ domain on the N-terminal side and two C2 @ domains on the C-terminal side like rabfilin. The central part has a PDZ {domain} structure (involved in the translocation of signal molecules into the intracellular membrane) and is localized in the active zone of the synapse. Although the detailed function of RIM is unknown, it is thought that it may be involved in synaptic vesicle binding to the synaptic active zone and in the release of neurotransmitters. Recently, a gene encoding a RIM binding protein has been cloned (J. Biol. Chem. 275, 26 (2000) 20030-20044), which has three SH3 domain and three fibronectin type III domain and SH3 It binds to the RIM via the domain. It is also thought that they attach to the matrix of the synaptic active zone and control the release of neurotransmitters.
[0077]
The protein of the present invention has 72% and 75% homology with RIM binding protein 2, respectively, and is therefore considered a RIM binding protein family molecule.
The binding between the protein of the present invention and RIM can be confirmed by a method known per se, for example, using a GST pull-down assay as follows (J. Biol. Chem. 275, 26 (2000) 2002003-20044). . First, rat brain is homogenized using a sample buffer containing 0.5% Triton X-100, 1 mM EDTA, 0.1 M NaCl, protease inhibitors, and 50 mM Hepes-NaOH (pH 7.4), and centrifuged to obtain whole brain. Obtain fractions. A fusion protein in which GST is linked to the N-terminus or C-terminus of the protein of the present invention is bound to glutathione agarose beads, and a brain homogenate treated with SDS to solubilize RIM is added thereto at 4 ° C. overnight. Incubate. After washing the beads with a sample buffer to remove non-specifically adsorbed substances, recovering the bound beads, performing SDS gel electrophoresis, and performing immunoblotting using an anti-RIM antibody, the protein of the present invention and RIM can be combined with each other. Can be examined for its specific binding ability.
[0078]
The study on the activity of releasing the neurotransmitter by the protein of the present invention can be carried out as follows. For example, a vector 1 encoding human growth hormone and a vector 2 incorporating a gene encoding the protein of the present invention or an empty vector 2 are transfected into PC12 cells, and after 3 days, depolarization stimulation with KCl is applied. By quantifying human growth hormone released into the medium, the release activity of the neurotransmitter can be examined. The confirmation of the activity of the protein of the present invention is not limited to these methods.
The above-described functional assay system can also be used for screening a function activator or a function inhibitor of the protein of the present invention described below, or a screening of a protein expression regulator of the present invention. Note that confirmation of the activity of the protein of the present invention is not limited to these methods.
[0079]
Examples of the method for analyzing the function of the protein of the present invention include, for example, (i) a method for comparative analysis of the expression state in each tissue, disease, or developmental stage; A method of analyzing, (iii) a method of introducing into a suitable cell or individual, and analyzing a phenotypic change, and (iv) a method of analyzing the phenotypic change by inhibiting expression of the protein in a suitable cell or individual And the like. Moreover, according to such a method, the activity specific to the target protein can be analyzed from many aspects.
[0080]
In the method (i), the expression of the protein of the present invention can be analyzed at the mRNA level or the protein level. When the expression level is analyzed at the mRNA level, for example, an in situ hybridization method (In situ hybridization: Application to Developmental Biology &Medicine; Medicine, Ed., by, Harry, N., Wid. 1990)), a hybridization method using a DNA chip, a quantitative PCR method, and the like. In the case of analysis at the protein level, ELISA using an antibody that specifically binds to the protein of the present invention described later, Western blotting, tissue staining, and the like can be mentioned. Here, when a known variant is present in the protein to be analyzed, it is preferable to use a probe that is present only in the cDNA encoding the protein to be analyzed and does not hybridize with the cDNA encoding the known variant. In the case of the quantitative PCR method, a method of selecting primers capable of producing an amplified fragment having a different length between the target cDNA and a known variant (Wong, {Y., Neuroscience Let., {320: 141-145} (2002)), etc. Is mentioned. Also, when analyzing at the protein level, it is preferable to use an antibody that reacts only with the target protein and does not react with a known variant.
[0081]
In the method (ii), the function of the protein of the present invention can be analyzed by examining the presence or absence of interaction between the protein of the present invention and a known protein. As a method for analyzing the interaction, a conventional method known per se can be used, and specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon method, phage display method, ribosomal display And the like. Also in this method, when a known variant exists in the protein to be analyzed, it is preferable to analyze the interacting substance of the known variant in the same manner to identify a substance that specifically interacts with the target protein.
[0082]
In the above method (iii), the cells into which the cDNA of the present invention is introduced are not particularly limited, but human cultured cells and the like are particularly preferably used. Methods for introducing DNA into cells include those described in (2) above. In addition, the phenotype of the introduced cells can be observed with a microscope, such as cell viability, cell growth rate, cell differentiation, neurite outgrowth when cells are neurons, localization and migration of intracellular proteins, etc. And those that can be analyzed by biochemical experiments, such as changes in the expression of specific proteins in cells. These phenotypes are associated with the target protein by introducing the DNA of the present invention or DNA encoding the known variant into cells in the same manner when a known variant exists in the target protein, and performing comparative analysis. The phenotype can be identified. In addition, since it is known that the protein of the present invention has RIM binding activity, it is also preferable to analyze by focusing on the phenotype and the like found in diseases associated with RIM binding activity.
[0083]
The method (iv) can be efficiently performed by a method using an oligonucleotide described below or an RNA interference method. In this method, when a known variant is present in the target protein to be analyzed, a similar analysis is performed for the known variant and other variants, and a target protein-specific function is identified by comparative analysis. be able to.
[0084]
(6) Screening for a molecule that regulates the activity of the protein of the present invention
By screening for a substance that specifically binds to the protein of the present invention and has an action of inhibiting, antagonizing, or enhancing the function (activity) of the protein of the present invention, a function modulator of the protein of the present invention (hereinafter, referred to as (Sometimes referred to as "modulators").
[0085]
This method of screening for a regulatory substance may be any method as long as it can obtain a substance that specifically binds to the protein of the present invention and has an activity of inhibiting, antagonizing or enhancing the activity of the protein. For example, first, the protein of the present invention is brought into contact with a test substance, and the test substance is selected based on the function of the protein of the present invention, that is, a change in RIM binding activity, after selecting the protein based on its binding to the protein. A selection method can be used.
[0086]
The test substance may be any substance as long as it interacts with the protein of the present invention and may affect the activity of the protein. , Proteins, non-peptidic compounds, low molecular weight compounds, synthetic compounds, fermentation products, cell extracts, animal tissue extracts and the like. These substances may be novel substances or known substances. As a method for analyzing the interaction between the test substance and the protein of the present invention, a conventional method known per se can be used. Specifically, for example, yeast two-hybrid method, fluorescence depolarization method, surface plasmon Method, a phage display method, a ribosomal display method, or a competition analysis method with the antibody described in the above (4). A substance found to bind to the protein of the present invention by such a method is then analyzed by analyzing how the activity of the protein of the present invention is affected in the presence of the substance. Whether it is used as a modulator is identified.
[0087]
Here, when using the DNA of the present invention or a recombinant protein used for screening a regulatory substance for the purpose of obtaining a pharmaceutically active ingredient, it is preferable to use the above-mentioned human homologous protein or orthologous protein. Further, the substance screened by the above method may be further selected as a drug candidate by screening in vivo.
[0088]
As described above, the protein having an RIM binding activity of the present invention has an important function as a regulatory factor involved in various physiological functions, and the abnormality of the protein in a living body causes various diseases. Therefore, modulators of RIM binding activity obtained by the above screening method are therapeutic agents for various diseases, for example, diseases related to abnormal neurotransmission, such as Alzheimer's dementia, Parkinson's disease, chorea, ischemic brain disease, It can be a therapeutic agent for diabetic peripheral neuropathy and the like.
[0089]
Such a modulator of RIM binding activity can be used alone for the clinical application, but can also be used as a pharmaceutical composition by mixing with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0090]
(7) Screening of the DNA expression regulator of the present invention
Examples of the screening method include a method of analyzing the expression level of the protein of the present invention or the mRNA encoding the same in the presence of a test substance. Specifically, for example, cells expressing the protein of the present invention described in (2) above are cultured in an appropriate medium containing a test substance, and the amount of the protein of the present invention expressed in the cells is determined by ELISA. And the like, or by analyzing the amount of mRNA encoding the protein of the present invention in the cells by quantitative reverse transcription PCR, Northern blotting, or the like.
[0091]
As the test substance, the substance described in the above (6) can be used. According to this analysis, if the amount of the protein or mRNA expressed in the cells cultured in the absence of the test substance increases as compared with the amount of the test substance, the substance functions as a substance for promoting the expression of the DNA of the present invention. If it is possible and, on the contrary, decreases, it can be determined that the substance can be used as a substance inhibiting the expression of the DNA of the present invention.
[0092]
The above-mentioned active ingredient can be used alone for clinical application, but can also be used as a pharmaceutical composition by blending it with a pharmaceutically acceptable carrier. At this time, the ratio of the active ingredient to the carrier can be varied between 1 and 90% by weight. Such drugs can be administered in various forms, such as tablets, capsules, granules, powders, orally administered by syrup or the like, or injections, drops, liposomes, Parenteral administration with suppositories and the like can be mentioned. In addition, the dose can be appropriately selected depending on symptoms, age, body weight, and the like.
[0093]
(8) The DNA-introduced animal of the present invention
The transfected DNA containing the DNA of the present invention described in the above (1) is constructed, introduced into a fertilized egg of a mammal other than a human, and this is transplanted into a female individual uterus to generate the DNA. A non-human mammal into which DNA has been introduced can be produced. More specifically, for example, after superovulation of a female individual by hormone administration, it is mated with a male, a fertilized egg is extracted from an oviduct on the first day after mating, and the introduced DNA is microinjected into the fertilized egg. It is introduced by the method described above. Thereafter, after culturing by an appropriate method, the surviving fertilized eggs are transplanted into the uterus of a pseudopregnant female individual (foster parent) to give birth. Whether or not the target DNA has been introduced into the newborn can be identified by performing Southern blot analysis on DNA extracted from cells of the individual. Examples of mammals other than humans include mice, rats, guinea pigs, hamsters, rabbits, goats, pigs, dogs, cats, and the like.
[0094]
The thus-obtained DNA-introduced animal of the present invention obtains its offspring by crossing this individual and subculturing it in a normal breeding environment while confirming that the introduced DNA is stably retained. be able to. In addition, the offspring can be obtained by repeating in vitro fertilization, and the strain can be maintained.
The non-human mammal into which the DNA of the present invention has been introduced can be used as an analysis of the function of the DNA of the present invention in a living body, or as a screening system for a substance regulating the function.
[0095]
(9) Other uses of the protein of the present invention and DNA containing a nucleotide sequence encoding the same
The protein of the present invention can be used as a carrier having it bound on a substrate. In addition, a nucleotide sequence encoding the protein of the present invention, for example, a DNA having the nucleotide sequence of SEQ ID NO: 1 and a partial fragment thereof can be used as a carrier obtained by binding them on a substrate. These may be hereinafter referred to as “protein chips”, “DNA chips” or “DNA arrays” (DNA microarrays and DNA macroarrays). These protein chips or DNA chips or arrays may contain other proteins and DNAs in addition to the proteins and DNAs of the present invention.
[0096]
Here, a resin substrate such as a nylon film or a polypropylene film, a nitrocellulose film, a glass plate, a silicon plate, or the like is used as a substrate for binding a protein or DNA. When using a fluorescent substance or the like, a glass plate or a silicon plate containing no fluorescent substance is preferably used. The binding of the protein or DNA to the base can be easily carried out by a commonly used method known per se. These protein chips, DNA chips, or DNA arrays are also included in the scope of the present invention.
[0097]
In addition, the amino acid sequence of the protein of the present invention and the nucleotide sequence of DNA can also be used as sequence information. Here, the nucleotide sequence of the DNA includes the nucleotide sequence of the corresponding RNA. That is, by storing the obtained amino acid sequence or base sequence in an appropriate recording medium in a predetermined format readable by a computer, a database of the amino acid sequence or base sequence can be constructed. This database may contain the base sequences of other types of proteins and DNAs encoding them. Further, in the present invention, the database also means a computer system that writes the above-mentioned sequence on an appropriate recording medium and performs a search according to a predetermined program. Examples of suitable recording media include magnetic media such as flexible disks, hard disks, and magnetic tapes; optical disks such as CD-ROM, MO, CD-R, CD-RW, DVD-R, and DVD-RW; and semiconductor memories. And the like.
[0098]
【Example】
Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.
Example 1 Preparation of cDNA library
(1) Preparation of mRNA
mRNA-prepared mouse (C57BL / 6) 0.5 to 1 g of each organ or tissue is homogenized with 10 ml of a suspension, and the same amount of 2 M {sodium acetate 1 ml} at pH 4.0 and the same amount of phenol / chloroform (5: 1 by volume). The mixture was added and extracted. When the same amount of isopropanol was added to the aqueous layer after the extraction, RNA separated and precipitated from the aqueous phase. After incubating the sample on ice for 1 hour, the precipitate was collected in a refrigerated centrifuge at 4,000 rpm for 15 minutes. The sample was washed with 70% ethanol, dissolved in 8 ml of water, and precipitated by adding 16 ml of an aqueous solution containing 2 ml of 5 M NaCl, 1% CTAB (cetyltrimethy-ｉlammonium bromide), 4 M urea, and 50 mM Tris, pH 7.0. To remove polysaccharide (CTAB precipitation).
[0099]
Subsequently, the RNA was centrifuged at 4,000 rpm for 15 minutes at room temperature to dissolve the RNA in 4 ml of 7M guanidine-C1. After adding twice the volume of ethanol, the mixture was incubated on ice for 1 hour, centrifuged at 4,000 rpm for 15 minutes, and the resulting precipitate was washed with 70% ethanol to collect RNA, which was dissolved again in water. RNA purity was measured by reading the OD ratios 260/280 (> 1.8) and 230/260 (<0.45).
[0100]
(2) Preparation of first strand cDNA
Using the mRNA prepared in the above (1) {15 μg, reverse transcriptase 3,000 units}, 5-methyl-dCTP, dATP, dTTP, and dGTP were converted to 0.54 mM and 0.6 M, respectively, in a reaction solution having a final volume of 165 μl. Trehalose, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂A reverse transcription reaction was performed under the conditions of 10 mM {DTT, 52 ng / μl} BSA, and 5 units of RNase inhibitor. 12.6 μl of an oligonucleotide (SEQ ID NO: 5) containing the recognition sequence of the restriction enzyme XhoI (wherein V represents A, G or C and N represents A, G, C or T) was used as a primer.
[0101]
At the start of the reaction, 1/4 of the reaction solution is collected, and 1.5 μl of [α-32P] -dGTP (3000 Ci / mmol, 10 μCi / μl; manufactured by Amersham) is added to the first strand cDNA. Was measured for synthesis efficiency. 0.5 μl of the RI-labeled reaction solution was spotted on DE-81 paper, and the RI activity before and after washing three times with 0.5 M sodium phosphate buffer (pH 7.0) was measured and calculated. Thereafter, the RI-labeled reaction solution and the non-labeled reaction solution were mixed, 0.5 M EDTA 8 μl, 10% SDS 2 μl, and proteinase K 20 μg were added, and the mixture was heated at 45 ° C. for 15 minutes. After extraction with phenol / chloroform and ethanol precipitation, the precipitate was dissolved in 47 μl of RNase-free water (hereinafter referred to as RNase-free water).
[0102]
(3) 5 'cap structure and addition of biotin to 3' end
Biotinylated RNA Diol In order to bind biotin to the diol site (present at both the 5 'end of the Cap structure and the ribose at the 3' end of the poly A chain), a two-step reaction was performed. They are the oxidation of the diol group followed by the coupling reaction of the biotin hydrazide with the oxidized RNA form. First, 15 μg of the RNA-first strand cDNA complex obtained by the reverse transcription reaction was placed in a 50 μl reaction mixture using a 6.6 mM sodium acetate buffer (pH 4.5) and sodium periodate as an oxidizing agent. Processed. This oxidation reaction was performed on ice for 45 minutes under light-shielded conditions.
[0103]
Subsequently, 11 μl of 5 M sodium chloride, 10 μl of SDS 0.5 μl, and the same amount of isopropanol were added, and the mixture was left on ice for 60 minutes, and centrifuged at 4 ° C. for 15 minutes at 15,000 rpm to obtain a precipitate. The precipitate was washed with 70% ethanol and redissolved in 50 μl of RNase-free water. To the sample, 5 µl of 1 M sodium acetate (pH 6.1), 5 µl of 10% SDS, and 150 µl of 10 mM biotin hydrazide (manufactured by Sigma) were added, and reacted at room temperature (22 to 26 ° C) overnight. Finally, 5 μl of 5M NaCl, 75 μl of 1 M sodium acetate (pH 6.1) and 2.5 volumes of ethanol were added, and the mixture was cooled on ice for 1 hour, centrifuged at 4 ° C. for 15 minutes, and biotinylated. After the reaction, the reaction solution was centrifuged for 15 minutes to precipitate the RNA-DNA complex again. The precipitate was washed once with 70% ethanol and once with 80% ethanol, and dissolved in 70 μl of RNase-free water.
[0104]
(4) Selection of full-length cDNA by RNase I
By treating the biotinylated RNA-DNA complex obtained in the above (3) with RNase I that digests single-stranded RNA, mRNA whose complete cDNA elongation was not obtained during the reverse transcription reaction, and mRNA The biotin residue labeled at the 3 'end was removed. Specifically, 10 μl of 10 × RNase I buffer (100 mM Tris-HCl (pH 7.5), 50 mM EDTA, 2 M NaOAc) was added to 70 μl of the sample obtained in (3), and RNase I (RNase One).^TM200 units (Promega), and the single-stranded RNA was digested at 37 ° C. for 15 minutes.
[0105]
(5) Collection of full-length cDNA
In order to prevent non-specific adsorption of cDNA to magnetic beads coated with streptavidin, 5 mg (500 μl) of magnetic beads (MPG) particles of 100 μg of yeast tRNA (treated with DNase I) were added. (CPG, NJ)) and left on ice for 1 hour, followed by washing with a solution of 50 mM EDTA and 2 M NaCl.
[0106]
The beads were suspended in 500 μl of a solution of 50 mM EDTA and 2 M NaCl, and the RNase I-treated cDNA obtained in (4) above was added. By stirring for 30 minutes at room temperature, the magnetic beads and the full-length cDNA were bound. The beads capturing the full-length cDNA were washed 4 times with a solution of 50 mM EDTA and 2 M NaCl, once with 0.4% SDS, 50 μg / μl yeast tRNA, 10 mM NaCl, 0.2 mM EDTA, and 10 mM Tris-HCl (pH 7.5). Once with 20% glycerol, once with a 50 μg / μl aqueous solution of yeast tRNA, RNase H buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2₂After washing once with 20 mM KCl, 0.1 mM EDTA, and 0.1 mM dithiothreitol (DTT), the cells were suspended in 100 μl of RNase H buffer, RNase H 3 units were added, and the mixture was heated at 37 ° C. for 30 minutes. Thereafter, 1 μl of 10% SDS and 2 μl of 0.5 M EDTA were added, the mixture was exposed to 65 ° C. for 10 minutes, and the supernatant was collected.
[0107]
The single-stranded full-length cDNA thus recovered was extracted with phenol / chloroform, and the volume of the solution was reduced to 100 μl or less using a speed bag, and then subjected to G25 / G100 @ Sephadex chromatography. The fraction having RI activity was collected in a silicon-treated microtube, and 2 μg of glycogen was added, and the precipitate obtained by ethanol precipitation was dissolved in 30 μl of ultrapure water.
[0108]
(6) Addition of oligo dG to single-stranded cDNA
30 μl of the single-stranded cDNA recovered in the above (5) was mixed with 200 mM sodium cacodylate (pH 6.9), 1 mM @MgCl in a final volume of 50 μl of the reaction solution.₂1 mM @ CoCl₂Under the conditions of 1 mM ３７2-mercaptoethanol and 100 μM dGTP, oligo dG addition reaction was carried out at 37 ° C. for 30 minutes using 32 units of terminal deoxynucleotidyl transferase (TaKaRa). At the end of the reaction, EDTA was added to 50 mM and dissolved in 31 μl of ultrapure water through a series of extractions with phenol / chloroform and ethanol precipitation.
[0109]
(7) Second strand cDNA synthesis
The synthesis of the second-strand cDNA using the first-strand cDNA as a template was performed as follows. In a reaction system having a final volume of 60 μl, a second strand low buffer (200 mM @ Tris-HCl (pH 8.75), 100 mM @ KCl, 100 mM (NH₄)₂SO₄, 20 mM MgSO4, 1% Triton X-100, 3 mg / μl BSA), 2nd strand high buffer (200 mM Tris-HCl (pH 9.2), 600 mM KCl, 20 mM MgCl₂) 3 μl, 0.25 mM each of dCTP, dATP, dTTP and dGTP, β-NADH 6 μl, 31 μl of first-strand cDNA with oligo dG added, 600 ng of second-strand primer-adapter (SEQ ID NO: 6), and Ex Taq DNA polymerase ( Second-strand cDNA was synthesized using 15 units of TaKaRa \ Ex \ Taq (TaKaRa), 150 units of thermostable DNA ligase (Ampligase; Epicentre), and 3 units of heat-resistant RNase \ H (Hybridase; Epicentre).
[0110]
The reaction was stopped by adding 1 μl of 0.5 M EDTA and further heating at 45 ° C. for 15 minutes in the presence of 1 μl of 10% SDS and 10 μg of proteinase (K) to dissolve the protein component. A double-stranded full-length cDNA purified by extraction with ethanol / chloroform and ethanol precipitation was obtained.
[0111]
(8) Preparation of library
The double-stranded full-length cDNA obtained by the above method was inserted into a λZAPIII vector and recovered as a library. The λZAPIII vector is a λZAPII (manufactured by STRATAGENE) vector in which a partial sequence (SEQ ID NO: 7) of the multicloning site is modified to SEQ ID NO: 8, and two SfiI sites are newly introduced.
[0112]
Further, a λPS (RIKEN) vector was prepared, and cDNA was inserted. λPS (RIKEN) (named λ-FLC-1 (FLC means FULL-LENGTHＴcDNA)) is a λPS vector of MoBiTec (Germany) modified for cDNA. That is, BamHI and SalI convenient for cDNA insertion are respectively introduced into cloning sites existing on both sides of 10 kbp ｋ stuffer, and a 6 kb DNA fragment is inserted into the XbaI site so that a cDNA of about 0.5 kb to about 13 kb can be cloned. (JP-A-2000-325080). Using this λ-FLC-1, for example, in the case of a lung cDNA library, the average chain length of the insert was 2.57 kb, and it was possible to actually clone an insert of 0.5 kb to 12 kb. In the case of the conventional method λZAP, the average chain length of the insert was 0.97 kb, indicating that the use of λ-FLC-1 enables the cloning of a large-sized cDNA more efficiently than λZAP.
[0113]
Example 2 Normalization / subtraction of full-length cDNA library
(1) Preparation of driver
The mRNA prepared in Example 1 (1) (hereinafter, this may be referred to as “(a) RNA driver”) and the RNA prepared by an in vitro transcription reaction were used as drivers. The latter RNA is further divided into two types (hereinafter referred to as “(b) RNA driver” and “(c) RNA driver”). One is obtained by recovering cDNA from RNA-cDNA removed by normalization and cloning into a phage vector. After infection with Escherichia coli, 1000 to 2000 plaques per one starting material are mixed to form one library (mini-library), which is converted into plasmid DNA by a conventional method (the phage is infected again with Escherichia coli together with helper phage to form a phagemid). , And another infection to obtain plasmid DNA).
[0114]
The obtained DNA was subjected to an in vitro transcription reaction (using T3 RNA polymerase or T7 RNA polymerase), treated with DNase I (RQ1-RNase free; manufactured by Promega) and Proteinase K, and then extracted with phenol / chloroform to obtain (b) RNA. Got a driver. At this time, as a starting material, a mini-library is prepared from each of nine types of tissues (pancreas, liver, lung, kidney, brain, spleen, testes, small intestine, stomach), and the nine types of mini-libraries are mixed. To obtain RNA. As another RNA, a library (about 20,000 clones) already stored as a non-overlapping clone is cultured, and the obtained DNA is subjected to an in vitro transcription reaction (b) in the same manner as an RNA driver (c). ) RNA driver.
[0115]
These three types of RNAs were labeled with biotinylated using Label-IT \ Biotin \ Labeling Kit (manufactured by Mirus Corporation), added to tester cDNA at a ratio of 1: 1: 1, and reacted with Rot10 (42 ° C). The second strand was synthesized with respect to the supernatant collected after the treatment with streptavidin beads (CPG).
[0116]
Example 3 Nucleotide sequencing of full-length cDNA clones
(1) clone rearray
One representative clone was selected from each cluster. Representative clones were selected by Q-bot (GENETIX @ LIMITED) and arrayed on a 384-well plate. At that time, E. coli was cultured in 50 μl of LB medium at 30 ° C. for 18 to 24 hours. At this time, when the cDNA library was introduced into the PS vector and transformed Escherichia coli DH10B, 100 mg / ml ampicillin and 50 mg / ml kanamycin were added, introduced into the Zap vector, and introduced into the SOLR system. If so, 100 mg / ml ampicillin and 25 mg / ml streptavidin were added.
[0117]
(2) Extraction of plasmid and InsSizing
Each of the clones cultured in the above (1) is further cultured in 1.3 ml of an HT solution containing 100 mg / ml ampicillin, and the cells are collected by centrifugation. Then, QIAprep @ 96 Turbo (manufactured by QIAGEN) is used. To recover and purify the plasmid DNA. In order to examine the chain length of the cDNA inserted in the obtained plasmid, 1/30 of the plasmid DNA obtained above was digested with the restriction enzyme PvuII and subjected to 1% agarose gel electrophoresis.
[0118]
(3) Sequencing
Three types of sequencers were used to analyze the full-length nucleotide sequence of the full-length cDNA inserted into the thus obtained plasmid. In addition, plasmids were divided into two categories: those having insertion sequences shorter than 2.5 kb and those having longer insertion sequences. Among these, the nucleotide sequence of the clone having an insertion sequence shorter than 2.5 kb was analyzed from both ends. At this time, the plasmid was prepared using the primers described in SEQ ID NO: 9 (sense strand) and 10 (antisense strand) when the vector was PS, and SEQ ID NO: 11 (sense strand) when the vector was Zap. And Primer Cycle Prime Sequencing Kit (manufactured by Amersham Pharmacia Biotech) using the primers described in Nos. 12 and 12 (antisense strand), and analyzed using Licor DNA4200 (long read sequencer).
[0119]
Gaps that could not be analyzed by the above nucleotide sequence analysis were determined by the primer walking method. At this time, ABI Prism 377 and / or ABI Prism 3700 (manufactured by Applied Biosystems Inc.), BigDye terminator kit, and Cycle Sequencing FS Ready Reaction Kit (Ambition, Inc.) were used.
[0120]
In addition, the sequence of a clone in which the inserted cDNA was longer than 2.5 kb was determined by the shotgun method. At that time, Shimadzu RISA 384 and DYEnic ET terminator cycle sequencing kit (manufactured by Amersham Pharmacia Biotech) were used. To generate a shotgun library, 48 DNA fragments grown by PCR from 48 independent representative clones were used. The end of the amplified DNA fragment was blunt-ended with T4 DNA polymerase.
This DNA fragment was inserted into a pUC18 vector, and Escherichia coli DH10B was transformed with the recombinant vector. A plasmid DNA was prepared from this Escherichia coli in the same manner as in the above (2).
[0121]
About those representative clones, the base sequence was determined by base sequence analysis from both ends, and the base sequences were ligated on a computer, and then subjected to sharing by Double, Stroke, Shearing, Device (manufactured by Fire Inc.). Nucleotide sequence determination by the shotgun method was performed with duplication of 12 to 15 clones. The gaps whose sequence could not be determined by this nucleotide sequence determination were determined by primer walking in the same manner as described above.
[0122]
Example 4 Analysis of nucleotide sequence
(1) dnaform43059 (SEQ ID NOS: 1, 3)
As shown in SEQ ID NO: 1, dnaform 43059 was composed of 3421 bases, and among them, base numbers 297 to 2507 were open reading frames {including a termination codon}. The amino acid sequence predicted from the open reading frame consists of 736 amino acid residues (SEQ ID NO: 3). A homology search was performed on the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 using BLAST. As a result, the SPTR protein database {(integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database)} contained (i) ) The database registration code HSAB2316, KIAA0318 @ gene, \ partial \ cds \ (Homo @ sapiens) was e-value: 0, with 75% identity over 725 amino acid residues, and (ii) the database registration code AY072908, RIM-binding @ prote. 2 (chicken) was hit at 72% identity over 722 amino acid residues at e-value: 0 and (iii) database entry number AF039957; eripheral benzodiazepine receptor interacting protein (Homo sapiens) is, e-value: 5 × 10⁻¹¹⁸In, a hit was obtained with 376 amino acid residues at 37% coincidence. From these results, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 was a kind of {RIM-binding} protein.
Since RIM-binding protein is considered to be involved in the secretion of neurotransmitters, expression control substances, function activators or function inhibitors of the protein of the present invention may be used in diseases involving abnormal neurotransmission, such as Alzheimer's type. There is a possibility that it can be developed as a therapeutic agent for dementia, Parkinson's disease, chorea, ischemic brain disease, diabetic peripheral neuropathy and the like.
[0123]
(2) dnaform50034 (SEQ ID NOs: 2 and 4)
As shown in SEQ ID NO: 2, dnform 50034 is composed of 4080 bases, of which base numbers 724 to 3939 constitute an open reading frame {including a stop codon}. The amino acid sequence predicted from the open reading frame consists of 1071 amino acid residues (SEQ ID NO: 4). When a homology search was performed using BLAST for the amino acid sequence encoded by the salt sequence shown in SEQ ID NO: 2, the SPTR protein database {(integrating the SWISS-PROT protein sequence database and the TrEMBL nucleic acid translation database)} contained (i) ) Database registration symbol HSAB2316, KIAA0318 {gene, {partial} cds} (Homo sapiens)} has e-value: 0, with 81% identity over 1077 amino acid residues, and (ii) database registration symbol AY072908, RIM-binding @ rote 2 (chicken) hits with 75% identity over 813 amino acid residues in e-value: 0, and (iii) database entry AF19933 , RIM binding protein 1A (Rbp1A) mRNA, partial cds (Rattus) is, e-value: 5 × 10^{-1 11}, With a 35% match over 843 amino acid residues. From these results, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 2 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 was one kind of RIM-binding @ protein.
[0124]
Since RIM-binding protein is considered to be involved in the secretion of neurotransmitters, expression control substances, function activators or function inhibitors of the protein of the present invention may be used in diseases involving abnormal neurotransmission, such as Alzheimer's type. There is a possibility that it can be developed as a therapeutic agent for dementia, Parkinson's disease, chorea, ischemic brain disease, diabetic peripheral neuropathy and the like.
[0125]
Example 5 Tissue expression analysis using PCR method
In order to examine changes in tissue expression of mRNA encoding the protein of the present invention in normal mice and diseased mice, PCR was performed according to a conventional method (Higuchi R, et al., Biotechnology, {11: {1026-30}) (1993). Was used to perform tissue expression analysis.
[0126]
(1) cDNA synthesis
Total RNA was extracted from 19 tissues of the following mice (Kazuo Moriwaki, one extra edition, Molecular Medicine Separate Volume, Vol. 36, “Spontaneous Disease Model Animals”, Nakayama Shoten, 1999), and reverse transcription was performed using oligo dT as a primer. CDNA synthesis was performed using the enzyme.
(A) Tissue of normal mouse and tissue of diabetic model mouse
{Circle around (1)} Control mouse C57BL / KsJ-、 + m / + m Jcl (female, 8 weeks old) whole brain, thalamus, lung, kidney, bone marrow, pancreas, fat cells, liver, eyes
(2) Diabetes model mouse C57BL / KsJ-db / db Jcl (female, 8-week-old) pancreas, fat cells, liver, eyes
(B) Tissue of senescence accelerating mouse
(1) Normal senescent mouse SAM R1 / TA Slc (male, 13 weeks old) hippocampus, frontal cortex
(2) Senescence-accelerated mouse: SAM P8 / Ta Slc (male, 15 weeks old) hippocampus, frontal cortex
(C) Tissue of mouse model of cancer metastasis
(1) Normal colon of control mouse Balb / c (female, 5 weeks old)
(2) Colon metastasis model mouse Balb / c (female, 6 weeks old) colon cancer (colon cancer cells Colon 26 are transplanted into the mouse abdominal cavity, and colon cancer is removed 2 weeks later)
[0127]
(2) Quantification by PCR method
The expression of the following two types of mRNA encoding the protein of the present invention is attached to the product using a light cycler quantitative PCR device (manufactured by Roche Diagnostics) and a LightCycler-FastStart DNA master SYBR Green I reagent. Quantification was performed according to the protocol provided. The synthetic DNA sequences used for quantitative PCR are shown below.
(A) dnaform43059
5'-side primer: (AGACACCGTGCTCTACCCTA) (SEQ ID NO: 13)
3'-side primer: (AGGCCATGCGTGTCTCTTT) (SEQ ID NO: 14)
(B) dnaform50034
5'-side primer: (CAAACAGCTTGCCAAAAGTTG) (SEQ ID NO: 15)
3'-side primer: (TTCTCGATCAGCGCCTTAGT) (SEQ ID NO: 16)
The quantification result was corrected using Glyceraldehyde @ 3-phosphate @ dehydrogenase (GAPDH) as an internal standard. That is, the expression amount (copy number / μl) of the target gene in each tissue was divided by the expression amount of GAPDH (copy number / μl) to obtain a constant (1 × 10⁶) (Note: 10 to the sixth power). Table 1 shows the results.
[0128]
[Table 1]

[0129]
As is clear from Table 1, the expression of dnaform43059 was high in the brain, increased in the diabetic pancreas, and decreased in colon cancer. Dnaform 50034 was highly expressed in the brain, increased in the diabetic pancreas, and decreased in colon cancer. From these results, it is considered that the protein encoded by the DNA of the present invention may be involved in a disease relating to a tissue in which the mRNA expression is varied as described above or a tissue having a large mRNA expression level. .
[0130]
That is, it was inferred from Example 4 that the protein encoded by dnaform43059 (SEQ ID NO: 1) was a kind of RIM-binding @ protein. RIM-binding @ protein is thought to be involved in secretion of neurotransmitters. From the above results, it was also found that the expression was high in the brain, increased in the diabetic pancreas, and decreased in colon cancer. From the above, the expression control substance, function activator, or function inhibitor of the protein of the present invention is a disease involved in abnormal neurotransmission, such as Alzheimer's dementia, Parkinson's disease, chorea, ischemic brain disease, and diabetic. It may be developed as a therapeutic agent for peripheral neuropathy. In addition, the protein of the present invention may be developed as a therapeutic agent for diabetes, cancer and the like.
[0131]
From Example 4, it was inferred from Example 4 that the protein encoded by dnaform50034 (SEQ ID NO: 2) was one kind of RIM-binding @ protein. RIM-binding @ protein is thought to be involved in secretion of neurotransmitters. From the above results, it was also found that the expression was high in the brain, increased in the diabetic pancreas, and decreased in colon cancer. From the above, the expression control substance, function activator, or function inhibitor of the protein of the present invention is a disease involved in abnormal neurotransmission, such as Alzheimer's dementia, Parkinson's disease, chorea, ischemic brain disease, and diabetic. It may be developed as a therapeutic agent for peripheral neuropathy. In addition, the protein of the present invention may be developed as a therapeutic agent for diabetes, cancer and the like.
[0132]
【The invention's effect】
Since the protein of the present invention has RIM binding activity, a substance that regulates the activity can be screened using the protein or a DNA encoding the protein, and a medicament that can act on diseases related to the protein or the like. It is useful for development of etc. In addition, it is considered that the DNA of the present invention and the protein encoded by the DNA can be applied to treatment and diagnosis of diabetes, cancer and the like.
This application is based on Japanese Patent Application (No. 2002-128807) filed on Apr. 30, 2002, the contents of which are incorporated herein by reference. The contents of the documents cited in the present specification are also incorporated herein by reference.
[0133]
[Sequence list]

Claims (15)

The following protein (a) or (b):
(A) a protein consisting of the amino acid sequence of SEQ ID NO: 3 or 4;
(B) 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: 3 or 4, and having RIM binding activity. A DNA encoding the protein according to claim 1. A full-length cDNA encoding the protein of claim 1. DNA of any of the following (a), (b) or (c):
(A) DNA having the nucleotide sequence of SEQ ID NO: 1 or 2.
(B) a DNA having a base sequence in which one or several bases are deleted, substituted and / or added in the base sequence of SEQ ID NO: 1 or 2, and which encodes a protein having RIM binding activity.
(C) a DNA having a base sequence capable of hybridizing under stringent conditions with a DNA having the base sequence of SEQ ID NO: 1 or 2 or a sequence complementary thereto and encoding a protein having RIM binding activity . A recombinant vector comprising the DNA according to claim 2. A gene-introduced cell into which the DNA according to any one of claims 2 to 4 or the recombinant vector according to claim 5 has been introduced, or an individual comprising the cell. A protein according to claim 1, which is produced by the cell according to claim 6. A sense oligonucleotide having the same sequence as 5 to 100 consecutive nucleotides in the base sequence of the DNA according to any one of claims 2 to 4, an antisense oligonucleotide having a sequence complementary to the sense oligonucleotide, and An oligonucleotide selected from the group consisting of oligonucleotide derivatives of sense or antisense oligonucleotides. An antibody or a partial fragment thereof that specifically binds to the protein according to claim 1. The antibody according to claim 9, wherein the antibody is a monoclonal antibody. The antibody according to claim 10, wherein the monoclonal antibody has an action of neutralizing the RIM binding activity of the protein according to claim 1 or 7. A method for screening for an activity-regulating substance of a protein, comprising bringing the protein according to claim 1 or 7 into contact with a test substance, and measuring a change in the activity of the protein due to the test substance. A method for screening a substance regulating the expression of a DNA, comprising bringing the test substance into contact with the gene-transfected cell according to claim 6, and detecting a change in the expression level of the DNA introduced into the cell. At least one or more amino acid sequence information selected from the amino acid sequence of the protein according to claim 1 and / or at least one or more nucleotide sequence selected from the nucleotide sequence of the DNA according to any one of claims 2 to 4. A computer-readable recording medium that stores information. A carrier to which the protein according to claim 1 and / or the DNA according to any one of claims 2 to 4 are bound.