JP2004229644A - New protein and dna encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein based on a physiological activity by analyzing base sequences of cDNA clones contained in a catalogued full-length cDNA library and specifying the physiological activity of the protein encoded with the cDNA clones for those having new sequences and to provide a method for utilizing the DNA encoding the protein. <P>SOLUTION: The protein is (a) a protein comprising a specific amino acid sequence or (b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and/or added in the specific amino acid sequence and having a binding activity to the TGFβ receptor family. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【０１３３】
表１から明らかなように、ｄｎａｆｏｒｍ５１８３９は同一クラスターに属するＦＡＮＴＯＭ ＮＯ：５４３０４１３Ｊ２２をプローブとして発現組織を検討すると、対照（１７．５日齢胎児全身）と比較して骨、１０日齢新生児皮膚、精巣、および、肺で発現が増加した。
【０１３４】
実施例６ 各完全長ｃＤＮＡがコードするタンパク質の総合解析
上記実施例４および実施例５の結果より、次のとおり、各完全長ｃＤＮＡがコードするタンパク質の総合解析を行った。
本発明のｃＤＮＡがコードするタンパク質は、キナーゼ欠損ヒトＴＧＦβ受容体スーパーファミリーサブユニットＡＦ３８７５１３と類似しており、対照（１７．５日齢胎児全身）と比較して骨、１０日齢新生児皮膚、精巣、および、肺で発現が増加した。
これらのことから、本タンパク質は、ＴＧＦβ受容体との結合活性を有するタンパク質であり、骨粗鬆症、自己免疫疾患などの免疫疾患・炎症疾患、癌、糸球体腎炎、神経の瘢痕形成、皮膚の瘢痕形成、眼の瘢痕形成、肺線維症、動脈傷害、増殖性網膜症、網膜剥離、呼吸促進症候群、肝硬変、ポスト心筋梗塞、ポスト脈管形成再狭窄、ケロイド瘢痕形成、強皮症、脈管障害、白内障、緑内障、及び不妊などに関連する機能を有し、これらの治療薬の開発に有用であると考えられた。
【０１３５】
【発明の効果】
本発明のタンパク質はＴＧＦβ受容体ファミリーとの結合活性等を有することから、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の開発に有用である。
本出願は、２００２年４月２３日付けの日本特許出願（特願２００２−１２０８５３）、および２００２年１２月４日付けの日本特許出願（特願２００２−３５２２７０）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。
【０１３６】
【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, oligo dT as a primer, and reverse transcription to an 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 existing only in such 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 Application Laid-Open No. 2000-325080; Carnini, P. et al. 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, but the physiological activity of the protein encoded by the cDNA is still unknown. Remains.
[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 has a binding activity with the TGFβ receptor family. A sequence specific to the protein was found, and the protein encoded by these cDNAs was identified as having a binding activity to the TGFβ receptor family. The expression levels of these cDNAs in each tissue were 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) The following protein of (a) or (b):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 3;
(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 sequences of SEQ ID NOs: 2 and 3, and which has a binding activity to the TGFβ receptor family.
[0010]
(2) A DNA encoding the protein of (1).
(3) A full-length cDNA encoding the protein according to (1).
(4) Any one of the following DNAs (a), (b) or (c):
(A) DNA having the nucleotide sequence of SEQ ID NO: 1.
(B) encoding a protein having a base sequence in which one or several bases have been deleted, substituted and / or added in the base sequence of SEQ ID NO: 1, and having a binding activity to a TGFβ receptor family; DNA.
(C) a protein having a nucleotide sequence capable of hybridizing under stringent conditions to a DNA having the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary thereto and having a binding activity to the TGFβ receptor family; DNA encoding.
[0011]
(5) A recombinant vector containing the DNA according to any of (2) to (4).
(6) A transgenic 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 one of (2) to (4), and an antisense having a sequence complementary to the sense oligonucleotide. An oligonucleotide selected from the group consisting of an 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 according to (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 binding activity of the protein according to (1) or (7) to the TGFβ receptor family.
[0014]
(12) 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 activity of the protein caused by the test substance. Screening method.
(13) A screening for a substance regulating the expression of a DNA, comprising bringing the test substance into contact with the gene-transfected 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 according to (1) and / or at least one selected from the nucleotide sequence of the DNA according to 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 (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 may be a protein comprising the amino acid sequence of SEQ ID NO: 2 or 3, or one or several amino acids 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 TGFβ receptor. Any substance can be used as long as it can encode a protein having a binding activity to a body family. Specifically, it may be only the translation region encoding the amino acid sequence or may include the full length of the cDNA.
[0016]
Specifically, as the DNA containing the full-length cDNA, for example, a DNA having the nucleotide sequence of SEQ ID NO: 1 and the like are mentioned. Examples of the translation region include those having the sequence shown in base numbers 218 to 869 of SEQ ID NO: 1. 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, but specifically, for example, can be obtained by the method described below. 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, a 5 ′ cap (^7MeG_pppN) A molecule serving as a tag is chemically bonded to a diol structure specific to a site, and reverse transcription is performed using this mRNA as a template and oligo dT as a primer. Then, only the full-length cDNA is separated using the function of the tag molecule. It is preferable to use the method (JP-A-9-248187; JP-A-10-127291). 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 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 full-length cDNA, the base sequence obtained from the sequence based on the terminal 100 is obtained by converting BLAST (http://www.ncbi.nlm.nih.gov/BLAST/; National 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 the nucleotide sequence of such a full-length cDNA include those having the nucleotide sequence of SEQ ID NO: 1. Examples of the translation region include those having the sequence shown in base numbers 218 to 869 of SEQ ID NO: 1.
[0021]
The thus obtained novel nucleotide sequence was subjected to homology search by BLAST (Basic local alignment search tool; Altschul, SF, et al., J. Mol. Biol., 215, 403-410 (1990)). protein feature search by HPMMPFAM, which is one of the functional groups of homology search and HMMER (sequence analysis method using hidden Markov model; Eddy, SR, Bioinformatics 14, 755-763 (1998)). //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 kinds of annotation information associated with hit sequences having sufficiently significant homology obtained as a result of the search. Here, a sufficiently significant hit sequence means that the degree of coincidence between the catalytic 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 many of the top hit catalytic domain sequences have been confirmed to have a binding activity to the TGFβ receptor family, the clones to be analyzed that are similar in sequence to the TGFβ receptor family also have the same function, that is, the TGFβ receptor family. The prediction holds that it will have a binding activity with.
[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 a function cannot be clarified by comparing the full length of one sequence to one sequence, if there is a characteristic region in the sequence, it can be found and its function can be predicted. . A specific example of the function prediction of the protein thus performed will be described below.
[0025]
The amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 was determined by Homo sapiens putative transmembrane protein NMA precursor by BLAST search to have an e-value of 3 × 10⁻⁷⁰With a 67% identity over 138 amino acid residues, the kinase-definitive TGFbeta superfamily receptor subunit was e-value: 1 × 10^-48, 67% identity over 153 amino acid residues, and Mus musculus, BMP active membrane-bound inhibitor, homolog (Xenopus laevis) e-value: 1 × 10^-48, 68% identity over 153 amino acid residues.
[0026]
From these facts, it can be inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 has binding activity to the TGFβ receptor family and is a kinase-specific TGFbeta superfamily receptor subunit.
The TGFβ receptor family is a receptor known to penetrate the cell membrane once and to have a serine threonine kinase region in the intracellular domain, and to classify it into type 1 and type 2 receptors based on structure and function. Can be. Type 1 receptors have a GS domain rich in glycine and serine N-terminal to the kinase region. When the TGFβ family ligand binds, the receptor forms a heterotetramer consisting of two molecules of type 1 receptor and two molecules of type 2 receptor, and the kinase of type 2 receptor causes the GS domain of type 1 receptor to form. Phosphorylation results in activation of type 1 receptor kinase and transmission of signals into cells. BAMBI (BMP and active membrane-bound inhibitor; Nature Vol. 401 480-483 (1999)), which is a pseudo-receptor of the TGFβ family reported recently, has a high homology with the type 1 receptor but a normal TGFβ receptor. The lack of a common serine threonine kinase domain in the family has prevented the transmission of signals by the TGFβ family.
[0027]
As described above, the protein of the present invention has high homology to the TGFβ family pseudo-receptors BAMBI and Nma (J. Dent. Res. 80 (10), 1895-1902 (2001)), and Since they do not have a common serine threonine kinase domain in the body family, they can be inferred to be pseudo-receptors of the TGFβ family.
[0028]
The DNA of the present invention may be obtained in a state in which there is a base deletion or insertion in the translation region. As a result of the above-described homology search and protein feature search, the base sequence of the DNA was determined. When deletion or insertion of bases in the base is estimated, a full-length cDNA without base deletion or insertion is obtained by a known method such as library screening or PCR cloning commonly used by those skilled in the art. be able to. The protein of the present invention is expressed using the full-length cDNA thus obtained, and can be used for functional analysis and the like.
[0029]
The DNA of the present invention thus obtained, whose base sequence is determined, and whose function is estimated, includes not only the base sequence of SEQ ID NO: 1 or the base sequence having the base sequence shown above as its translation region, but also 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 bases and having a binding activity to the TGFβ receptor family; DNAs that hybridize with these under stringent conditions and encode a protein having a binding activity to the TGFβ receptor family, etc. included. As described above, these DNAs comprise an amino acid sequence in which one or several amino acid sequences have been deleted, substituted and / or added in the amino acid sequence of the protein shown in SEQ ID NOs: 2 and 3, and have a TGFβ receptor family. And those encoding proteins having a binding activity.
[0030]
Here, a DNA that hybridizes under stringent conditions means a homology of 80% or more, preferably 90% or more, more preferably 95% or more with the base sequence shown in SEQ ID NO: 1 or its complementary sequence by BLAST analysis. And a DNA containing a base sequence having the formula: 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.
[0031]
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).
[0032]
The nucleotide sequence of SEQ ID NO: 1 is derived from a mouse. A human cDNA library was prepared according to the above-described method for preparing a cDNA library, and the nucleotide sequence of SEQ ID NO: 1 was compared to the library. By performing hybridization using a DNA fragment having a nucleotide sequence as a probe, a DNA encoding a human homolog protein of the protein encoded by the nucleotide sequence of SEQ ID NO: 1 can also be obtained. The DNA that hybridizes under stringent conditions with the DNA of SEQ ID NO: 1 of the present invention also includes DNA encoding such a human homolog.
[0033]
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.
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, Database (including cDNA database predicted by informatics), a method of performing homology search using BLAST or the like, and (ii) using a base sequence of the target cDNA as a query, A method in which a homology search is performed using BLAST or the like, and the sequence of the hit EST is linked with reference to the base sequence of the target cDNA, and (iii) the base sequence of the target cDNA is used as a query, and Homology search using BLAST etc. against the genome database of Then, the position on the genome where the gene of the cDNA of interest is present 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 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 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, so the method described in (ii) or (iii) is preferably used.
[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 can be obtained from the above human cDNA library. As a specific acquisition method, for example, 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, PCR is performed using the above human cDNA library as a template. 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. (1) First, the homology between the nucleotide sequence of the cDNA of the present invention and the nucleotide sequence of the obtained human homolog DNA is analyzed. Next, using the base sequence of the cDNA of the present invention as a query, a homology search was performed for human base sequences contained in international base sequence databases such as DDBJ, EMBL, and GenBank, and patent databases. Confirm that the degree of matching of the base sequence is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query. Further, (2) homology is analyzed between the obtained nucleotide sequence of the human homolog DNA and the corresponding nucleotide sequence of the cDNA of the present invention. Next, using the base sequence of the obtained human homolog 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, and GenBank, and in the patent database. Confirm that the degree of matching of the base sequence is higher than the degree of matching between the base sequence obtained from the database and the base sequence of the query. By confirming the above (1) and (2), the obtained human homolog can be identified as a human ortholog corresponding to the cDNA of the present invention. The homology analysis described in (1) and (2) 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 (1) and (2) 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 thus obtained human homologous DNA or orthologous DNA, the function of the protein encoded by the nucleotide sequence can be estimated and confirmed.
The DNA that hybridizes under stringent conditions with the DNA having the base sequence of SEQ ID NO: 1 or its complementary sequence also includes DNA encoding such a human homolog or ortholog protein.
[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. Such amino acid sequences include, for example, those described in SEQ ID NOs: 2 and 3. 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 a TGFβ receptor. Those having a binding activity with the family are also included.
[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 (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 this recombinant vector is used to transform a suitable host microorganism or introduced into a suitable cultured cell. Thus, it can be obtained by purifying this.
[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 a binding activity to the TGFβ receptor family described above.
[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 (Stratagene) and the like. In the case of insect cells, BacPAK6 (Clontech) and the like are used. When the host is an animal cell, ZAP Express (manufactured by Stratagene), pSVK3 (manufactured by Amersham Pharmacia Biotech) and the like can be mentioned.
[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. For example, when the host is Escherichia coli, T3, T7, tac, A lac promoter or the like can be used. In the case of yeast, an nmt1 promoter, a Gal1 promoter, or 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) are used for E. coli, SP-Q01 (manufactured by Stratagene) is used for yeast, and AcNPV is used for baculovirus (J. Biol. Chem., 263, 7406, ( 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)). From the expression level and simplicity of screening, African green monkey kidney-derived COS-7 (ATCC CRL1651: American Type Culture Collection preserved cells), human fetal kidney-derived HEK293 cells (ATCC CRL1573), human cervical cancer HeLa cells (ATCC CCL-2) and the like are preferably used.
[0048]
In addition to the above-described expression method using a protein expression vector, 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 a 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 in 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, of course, included in the protein of the present invention as long as it has a binding activity to the TGFβ receptor family, 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 and functional analysis using the 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 described in 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-propyl ribose, or ribose in the oligonucleotide is 2 Oligonucleotide derivatives substituted with '-methoxyethoxyribose can be mentioned.
[0056]
The oligonucleotide of the present invention can be applied to the RNA interference method (hereinafter, this may be referred to as “RNAi method”) by preparing it as double-stranded RNA. For the method for producing double-stranded RNA and the RNA interference method, for example, the method described in (Elbashir, S., et al., Nature, 411, 494-498 (2001)) can be used. .
The double-stranded RNA does not need to be all RNA. Specifically, as a part of which is DNA, 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 polynucleotide consisting of RNA having 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 a target gene. And a sequence substantially identical to a sequence of 15 bp or more, which may be any part of the base sequence. 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, PI, et al., Interferon, 5, 115-180 (1983)), and when the double-stranded RNA enters a cell, PKR (dsRNA-responsive) is obtained. Protein kinase: Non-specifically inhibits the initiation of translation of many genes via Bass, BL, Nature, 411, 428-429 (2001)), and at the same time, 2 ', 5' oligoadenylate synthetase (Bass, B.L., Nature, 411, 428-429 (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. Examples of the method of association include a method in which the above polynucleotides are 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 by industry, and are pathogenic to 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 the 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, 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]
In addition, a monoclonal antibody can 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 or 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]
When a human-derived protein is obtained as the protein of the present invention, the above-described method is applied to a Severe combined immunodeficiency (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 immunization in the same manner as described above and preparing a hybridoma of the antibody-producing cells of the immunized animal and human myeloma cells (Mosier, DE, et al. Nature, 335, 256-259 (1988); Duchosal, MA, 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 thus-obtained antibody that specifically binds to the protein of the present invention can also be used as a neutralizing antibody that specifically binds to the protein of the present invention to thereby inhibit the binding activity of the protein to the TGFβ receptor family. it can. 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 and analysis of the activity 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 (1). Furthermore, analysis can also be performed by combination with the antibody or the like prepared as described in (4) above.
[0076]
As described in the above (1), the protein of the present invention can be assumed to be a pseudo-receptor of the TGFβ family. Such a binding activity between the protein of the present invention and the TGFβ receptor family can be assayed, for example, by those skilled in the art as follows.
[0077]
For example, a clone in which an HA tag is bound to the C-terminus of the TGFβ receptor family and a clone in which a flag tag is bound to the C-terminus of the protein of the present invention are produced, co-expressed in cos1 cells and the like, and TGFβ family molecules are acted on. After that, the cell lysate was obtained, immunoprecipitated using an anti-flag antibody, subjected to electrophoresis, and subjected to Western blot using an anti-HA antibody to confirm the binding between both proteins (Nature Vol. 401, 480-483 (1999)).
[0078]
The effect of the protein of the present invention on signal transduction by TGFβ family molecules can be evaluated, for example, by an assay system using a reporter gene having a BMP response element (BRE) or an activin response element (ARE). Specifically, using a reporter gene in which a luciferase gene is bound downstream of the BRE sequence, the protein of the present invention, the TGFβ receptor family, and the reporter gene are co-expressed in P19 cells or frog oocytes and the like, and TGFβ family molecules are expressed. The biological activity can be assayed by measuring the amount of luminescence when acted.
[0079]
Note that confirmation of the activity of the protein of the present invention is not limited to the method described above. In addition, these functional assay systems can also be used for screening of a function activator or a function inhibitor of the protein of the present invention described later, and a screening of a protein expression regulator of the present invention.
[0080]
In general, the method for analyzing the function of the protein of the present invention includes, for example, (i) a method for comparatively analyzing the expression state of each tissue, disease, or developmental stage, and (ii) an interaction with another protein or DNA. 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 the expression of the protein in a suitable cell or individual. And the like. According to such a method, the activity specific to the target protein can be analyzed from multiple aspects.
[0081]
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., Ed. by Harry, N. ed. 1990)), a hybridization method using a DNA chip, a quantitative PCR method, and the like. In the case of analysis at the protein level, a tissue staining method using an antibody that specifically binds to the protein of the present invention described later, an ELISA method, a Western blot method, and the like can be mentioned. Here, when the protein to be analyzed is a splicing variant in which a known variant is present, 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 should be used. Is preferred. In the case of the quantitative PCR method, a method of selecting and performing a primer capable of producing an amplified fragment having a different length between the target variant and the 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.
[0082]
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. In the method, when the protein to be analyzed is a splicing variant in which a known variant is present, the known variant is similarly analyzed for interacting substances, and a substance that specifically interacts with the target protein is identified. Is preferred.
[0083]
In the 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. In the case of a splicing variant in which a known variant exists, these phenotypes can be similarly introduced into cells, and a phenotype related to the variant to be analyzed can be identified by comparative analysis. Since the protein of the present invention is known to have TGFβ receptor binding activity, attention should be paid to phenotypes and the like found in diseases associated with TGFβ receptor binding activity. Analysis is also preferred.
[0084]
The method (iv) can be efficiently performed by a method using an oligonucleotide described below or an RNA interference method. In this method, if 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 function specific to the target protein is identified by comparative analysis. Can be.
[0085]
(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").
[0086]
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, a method of first contacting a protein of the present invention with a test substance and selecting the test substance based on the binding property to the protein as an index, and then selecting a test substance using the change in the activity of the protein of the present invention as an index. Can be used.
[0087]
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 Phage display method, ribosomal display method, or the 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.
[0088]
Here, for the purpose of screening for a pharmaceutically active ingredient, it is preferable to use the above-mentioned human homologous protein or orthologous protein for the DNA or recombinant protein of the present invention to be used. Furthermore, the substances screened by the above method may be selected as drug candidates by screening in vivo.
[0089]
The analysis of the change in the activity of the protein of the present invention can be carried out based on the above-mentioned method for analyzing the function of the protein of the present invention (a method for confirming the activity) by a method known per se and used in the art. Hereinafter, a method for analyzing a substance that regulates each activity of the protein of the present invention will be described with reference to specific examples.
[0090]
As a method for analyzing a substance that regulates the binding activity to the TGFβ receptor family, for example, a protein serving as a substrate is introduced into the above-described DNA transductant by the same method. The dephosphorylation of the protein serving as a substrate in the presence / absence of the selected substance with respect to this transductant is analyzed by a commonly used method known per se. Specifically, it can be performed using the method described in (5) above. When the binding activity to the TGFβ receptor family is increased as compared to the absence of the substance, the substance may function as a TGFβ receptor family binding activator, and may decrease, Alternatively, when inhibited, it can be identified that the substance may function as an inhibitor of the binding activity to the TGFβ receptor family.
[0091]
Although the protein of the present invention has a binding activity to the TGFβ receptor family, the TGFβ receptor family is involved in fibrosis of tissues related to, for example, renal fibrosis, pulmonary fibrosis, myocardial infarction and the like. Thus, the compounds identified as the receptor expression modulators and the like by the present screening method can be used as therapeutic agents for various fibrotic diseases and the like.
[0092]
Specific examples of the type of disease include glomerulonephritis, scarring of nerves, scarring of skin, scarring of eyes, pulmonary fibrosis, arterial injury, proliferative retinopathy, retinal detachment, and respiratory distress syndrome. Cirrhosis, post-myocardial infarction, post-angioplasty restenosis, keloid scar formation, scleroderma, vascular disorders, cataract, glaucoma, osteoporosis and the like. The therapeutic agent for such a disease is for treating a condition in which the effect of the TGFβ family molecule is harmful to an individual, and is characterized in that the effect is an effect of promoting fibrosis.
[0093]
Such modulators can be used alone for the clinical application, but can also be used as pharmaceutical compositions 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.
[0094]
(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) 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 or 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.
[0095]
As the test substance, those described in (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.
[0096]
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.
[0097]
(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. And so on. 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.
[0098]
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.
[0099]
(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. Further, 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 to which they are bound 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.
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 proteins and 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.
[0100]
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, a database of amino acid sequences and nucleotide sequences can be constructed by storing the obtained amino acid sequences and nucleotide sequences in an appropriate recording medium in a computer-readable predetermined format. 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.
[0101]
【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 1 ml of 2 M sodium acetate 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 added with 2 ml of 5 M NaCl, 1% of CTAB (cetyltrimethy-lammonium bromide), 4 ml of urea, and 16 ml of an aqueous solution of pH 7.0 containing 50 mM Tris to remove RNA. The precipitate was removed to remove the polysaccharide (CTAB precipitation).
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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).
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(2) Preparation of first strand cDNA
Using 15 μg of the mRNA prepared in (1) above, 5-methyl-dCTP, dATP, dTTP, and dGTP were converted to 0.54 mM and 0.6 M trehalose in a reaction volume of 165 μl using reverse transcriptase 3,000 units. The reverse transcription reaction was performed under the following conditions: 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2, 10 mM DTT, 52 ng / μl BSA, and RNase inhibitor 5 units. An oligonucleotide containing a recognition sequence of a restriction enzyme XhoI (SEQ ID NO: 4) (in the sequence, V represents A, G, or C, and N represents A, G, C, or T) 12.6 μl used as a primer Was.
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At the start of the reaction, 1/4 of the reaction solution is collected and 1.5 μl of [α-³²By adding P] -dGTP (3000 Ci / mmol, 10 μCi / μl; manufactured by Amersham), the synthesis efficiency of the first strand cDNA was measured. 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, 8 μl of 0.5 M EDTA, 2 μl of 10% SDS and 20 μg of proteinase K 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).
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(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 a diol group followed by the coupling reaction of biotin hydrazide with an oxidized RNA. 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 under light-shielding conditions for 45 minutes.
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Subsequently, 11 μl of 5 M sodium chloride, 0.5 μl of 10% SDS, and the same amount of isopropanol were added, 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. 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 to the sample, and reacted overnight at room temperature (22 to 26 ° C.). Finally, 5 μl of 5 M 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.
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(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 mRNA was not completely elongated during the reverse transcription reaction, and mRNA of 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.
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(5) Collection of full-length cDNA
In order to prevent non-specific adsorption of cDNA to magnetic beads coated with streptavidin, 5 μg (500 μl) of 100 μg of yeast tRNA (treated with DNase I) was used as magnetic beads (MPG) particles coated with streptomyvit. (CPG, NJ)) and left on ice for 1 hour, followed by washing with a solution of 50 mM EDTA and 2 M NaCl.
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These 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) 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, and 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 2₂, 20 mM KCl, 0.1 mM EDTA, 0.1 mM dithiothreitol (DTT)), suspended in 100 μl of RNase H buffer, added with 3 units of RNase H, and 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.
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The thus recovered single-stranded full-length cDNA 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.
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(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 2 in a final volume of 50 μl of the reaction solution.₂, 1 mM CoCl₂Under the conditions of 1 mM 2-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.
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(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 MgSO₄3%, 1% Triton X-100, 1 mg / μl BSA, second 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, 6 μl of β-NADH, 31 μl of first-strand cDNA to which oligo dG was added, 600 ng of second-strand primer-adapter (SEQ ID NO: 5), 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).
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The reaction was stopped by adding 1 μl of 0.5 M EDTA, and further heated at 45 ° C. for 15 minutes in the presence of 1 μl of 10% SDS and 10 μg of proteinase K to finally dissolve the protein components. A double-stranded full-length cDNA purified by extraction with ethanol / chloroform and ethanol precipitation was obtained.
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(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 obtained by modifying SEQ ID NO: 6, which is a partial sequence of the multiple cloning site, to SEQ ID NO: 7, and newly introducing two SfiI sites.
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Further, a λPS (RIKEN) vector was prepared, and cDNA was inserted. λPS (RIKEN) (named λ-FLC-1 (FLC means FULL-LENGTH cDNA)) 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 a 10 kbp stuffer, and a 6 kb DNA fragment is inserted into an 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.
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Example 2  Normalization / subtraction of full-length cDNA library
(1) Preparation of driver
The mRNA prepared in Example 1 (1) (hereinafter sometimes referred to as “(a) RNA driver”) and the RNA prepared by 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 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).
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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 RNA (b). An RNA driver was obtained. 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 in vitro transcription reaction in the same manner as (b) RNA driver (c). ) RNA driver.
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These three kinds of RNAs were labeled with biotin 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).
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Example 3  Nucleotide sequencing of full-length cDNA clones
(1) clone rearray
One representative clone was selected from each cluster. Representative clones were selected with 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.
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(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 of 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.
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(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: 8 (sense strand) and 9 (antisense strand) when the vector was PS, and SEQ ID NO: 10 (sense strand) when the vector was Zap. And a primer described in 11 (antisense strand) and reacted with a Thermosequenase Primer Cycle Sequencing Kit (manufactured by Amersham Pharmacia Biotech), and analyzed using a Licor DNA4200 (long read sequencer).
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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 (Applied Systems, Inc.) were used.
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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 DYEnamic 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 ends of the amplified DNA fragment were 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 was prepared from this E. coli in the same manner as in the above (2).
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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 using Double Stroke Sharing 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.
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Example 4  Analysis of nucleotide sequence
As shown in SEQ ID NO: 1, dnaform 51839 is composed of 1156 bases. A homology search was performed on the amino acid sequence encoded by SEQ ID NO: 1 using BLAST, and the result was that (i) a database registration symbol was found in the SPTR protein database (integrated SWISS-PROT protein sequence database and TrEMBL nucleic acid translation database). AJ277487, Homo sapiens putative transmembrane protein NMA precursor, e-value: 3 × 10⁻⁷⁰And 67% identity over 138 amino acid residues, and (ii) the database registration number AF383513, kinase-specific TGFbeta superfamily receptor subunit, e-value: 1 × 10^-48And 153 amino acid residues with 67% identity, and (iii) the database registration symbols BC019378, Mus musculus, BMP and active membrane-bound inhibitor, homolog (Xenopus laevis), e-value: 1 × 10^-48153 amino acid residues with 68% match. From these results, it was inferred that the protein encoded by the nucleotide sequence shown in SEQ ID NO: 1 had a binding activity to a TGFβ receptor and was a kinase-specific TGFbeta superfamily receptor subunit.
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Homology of (i) and (ii) above to the amino acid sequence (SEQ ID NO: 2) encoded by nucleotides 223 to 444 of SEQ ID NO: 1 and the amino acid sequence encoded by nucleotides 450 to 869 (SEQ ID NO: 3) Was seen. It is thought that the protein encoded by this gene is translated from the region of base numbers 218 to 869 of SEQ ID NO: 1. It was thought that there was a base insertion.
The TGFβ receptor family is related to bone formation, autoimmune diseases, renal fibrosis, pulmonary fibrosis, myocardial infarction, etc., and activators or inhibitors of this receptor are used as therapeutic agents for these diseases. It was speculated that it could be developed.
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Example 5  Tissue expression analysis using DNA microarray
Tissue expression analysis is described in Miki, R .; , Et al. , Proc. Natl. Acad. Sci. USA, 98, 2199-2204 (2001).
(1) Preparation of DNA microarray
One type of mouse cDNA library FANTOM (HYPERLINK http://fantom.gsc.liken.go.jp/http://) belonging to the same cluster as the mouse full-length cDNA to be analyzed (having a base sequence homologous to the cDNA). After amplifying the nucleotide sequence (FANTOM NO: 5430413J22) of the cDNA derived from phantom.gsc.liken.go.jp/) using M13 forward and reverse primers, the PCR product was precipitated with isopropanol and 15 μl of 3 × SSC Dissolved in the liquid. This one type of DNA solution was spotted on a glass slide coated with poly-L-lysine using a DNA arrayer of 16 chips (SMP3, TeleChem International, Sunnyvale, CA) to prepare a DNA microarray (for details of the method, see http: //Cmgm.stanford.edu/pbrown/mgide/index.html). Mouse β-actin and glyceraldehyde-3-phosphate dehydrogenase cDNA were used as a positive control, and Arabidopsis thaliana cDNA was used as a negative control.
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The detection sensitivity of this DNA microarray was 1 to 3 copies of mRNA per cell. The signal intensity of clones having approximately 80% identity with the target sequence was one-tenth that of clones with perfect sequence identity. The signal intensity of clones with less than 80% match with the target sequence was at the background level.
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(2) Preparation of probe
22 tissues of fetal, neonatal and adult C57BL / 6J mice (kidney, brain, spleen, lung, liver, testis, pancreas, stomach, small intestine, colon, placenta, heart, thymus, cerebellum, uterus, bone, muscle, spine 1 μg of mRNA extracted from side kidney-derived adipocytes, epididymis-derived adipocytes, visceral fat, 10-day-old neonatal cerebellum, 10-day-old neonatal skin) was subjected to a random prime reverse transcription reaction according to a conventional method, and a fluorescent dye Cy3 (manufactured by Amersham Pharmacia) was used. I took in. On the other hand, 1 μg of mRNA extracted from the whole body of a 17.5-day-old fetus was subjected to a random prime reverse transcription reaction, and the fluorescent dye Cy5 was taken in as a control for expression analysis. The CyDye-labeled cDNA probe was purified using CyScribe GFX Purification Kit (manufactured by Amersham Pharmacia) and eluted from the column with 17 μl of sterilized water. To this was added a blocking solution consisting of 3 μl of 10 μg / μl oligo (dA), 3 μl of yeast tRNA 20 μg / μl, 1 μl of 20 μg / μl mouse Cot1 DNA, 5.1 μl of 20 × SSC, and 0.9 μl of 10% SDS. By mixing, a CyDye-labeled cDNA probe was prepared.
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(3) Hybridization of DNA microarray
30 μl of a solution in which a cDNA probe (Cy3 label) derived from the tissue to be analyzed for expression and a control 17.5 day-old fetal cDNA probe (Cy5 label) were mixed was heat-treated at 95 ° C. for 1 minute, and cooled at room temperature. The probe solution was added to the DNA microarray, covered with a cover slip, and hybridized at 65 ° C. overnight in Hybridasette (manufactured by ArrayIt). Next, the DNA microarray was washed with 2 × SSC, 0.1% SDS, and subsequently rinsed with 1 × SSC for 2 minutes and 0.1 × SSC for 2 minutes. The microarray was scanned using a ScanArray 5000 confocal laser scanner, and the image was analyzed with IMAGEENE (manufactured by BioDiscovery).
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(4) Data analysis
The amount of mRNA (Cy3-labeled) in each tissue is expressed as the ratio (Cy3 / Cy5) to the amount of fetal whole-body mRNA at 17.5 days of age (Cy5-labeled) as a logarithm (log).₂). Experiments were performed twice independently to increase the accuracy of the data and reproducible results were employed. The results are shown in Table 1 below.
Generally, in expression analysis using a DNA array, an increase or decrease of about 2 times is regarded as an experimental error. From this, when the numerical value of the result shown in Table 1 is 1 or more, the amount of mRNA in a certain tissue is more than twice as large as the amount of mRNA of the whole body of a fetal 17.5-day-old control, which is significant. Was interpreted as increasing. Conversely, when the numerical value of the result is -1 or less, the amount of mRNA in a certain tissue is less than one-half that of the whole fetal mRNA at 17.5 days of age as a control, and is significantly reduced. I interpreted that. When comparing the mRNA expression levels between any tissues, if the difference between the values in each tissue is 1, the mRNA level is 2 times, and if it is 2, the mRNA level is 4 times. If the difference between the numerical values is -1, the amount of mRNA is 1/2 times, and if it is -2, the amount of mRNA is 1/4 times.
Note that dnaform 51839 belongs to the same cluster as the DNA spotted on the microarray, and is a mouse cDNA clone having a region having a nucleotide sequence identity of at least 80% or more over at least 200 bases. The results were described as cDNA, and the values of the measurement results of the DNA spotted on the microarray were described as substitutes.
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[Table 1]

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As is clear from Table 1, when dnform 51839 belongs to the same cluster, and FANTOM NO: 5430413J22 is used as a probe, the expression tissues are examined. And increased expression in lung.
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Example 6  Comprehensive analysis of proteins encoded by each full-length cDNA
Based on the results of Example 4 and Example 5, comprehensive analysis of the protein encoded by each full-length cDNA was performed as follows.
The protein encoded by the cDNA of the present invention is similar to the kinase-deficient human TGFβ receptor superfamily subunit AF385513, and compared to the control (17.5-day-old fetal whole body), bone, 10-day-old neonatal skin, testis And increased expression in lung.
From these facts, the present protein is a protein having a binding activity to TGFβ receptor, and is used for immunological diseases and inflammatory diseases such as osteoporosis and autoimmune diseases, cancer, glomerulonephritis, nerve scar formation, and skin scar formation. Ocular scar formation, pulmonary fibrosis, arterial injury, proliferative retinopathy, retinal detachment, respiratory distress syndrome, cirrhosis, post-myocardial infarction, post-angioplasty restenosis, keloid scar formation, scleroderma, vascular disorders, It has functions related to cataract, glaucoma, infertility and the like, and was considered to be useful for the development of these therapeutic agents.
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【The invention's effect】
Since the protein of the present invention has a binding activity to the TGFβ receptor family and the like, a substance that regulates the activity can be screened using the protein or a DNA encoding the protein, and diseases associated with the protein can be screened. It is useful for the development of a medicament capable of acting on the skin.
This application is based on a Japanese patent application filed on Apr. 23, 2002 (Japanese Patent Application No. 2002-120853) and a Japanese patent application filed on Dec. 4, 2002 (Japanese Patent Application No. 2002-352270). Is hereby incorporated by reference. The contents of the documents cited in the present specification are also incorporated herein by reference.
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[Sequence list]

Claims (15)

The following protein (a) or (b):
(A) a protein comprising the amino acid sequence of SEQ ID NO: 2 or 3;
(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 sequences of SEQ ID NOs: 2 and 3, and which has a binding activity to the TGFβ receptor family. A DNA encoding the protein according to claim 1. A full-length cDNA encoding the protein according to claim 1. The DNA according to any one of the following (a), (b) or (c):
(A) DNA having the nucleotide sequence of SEQ ID NO: 1.
(B) encoding a protein having a base sequence in which one or several bases have been deleted, substituted and / or added in the base sequence of SEQ ID NO: 1, and having a binding activity to a TGFβ receptor family; DNA.
(C) a protein having a nucleotide sequence capable of hybridizing under stringent conditions to a DNA having the nucleotide sequence of SEQ ID NO: 1 or a sequence complementary thereto and having a binding activity to the TGFβ receptor family; DNA encoding. 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 the sense or antisense oligonucleotide. 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 binding activity of the protein according to claim 1 or 7 to the TGFβ receptor family. 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.