JP2005046024A - New protein kinase and gene encoding protein kinase splice mutant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new protein belonging to protein kinase, and to provide a gene encoding a splice mutant. <P>SOLUTION: This DNA containing a base sequence encoding the following protein (a) or (b). (a) A protein comprising the same or substantially same amino acid as a specific amino acid sequence. (b) A protein comprising the above-mentioned amino acid sequence whose amino acids are partially deleted, substituted or added and having the substantially same biological activity as the protein kinase. A recombinant encoded in the DNA. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【０１３１】
Ｃ−ＢＲＡＣＥ２０４７５７３のクロモゾーム上の位置を特定するため配列番号：１をクエリーとしてヒトゲノム配列データベース（Ａｐｒ．１４，２００３）に対してＢＬＡＳＴ検索を実施した。得られた結果を通常期待されるエクソン−イントロン境界（ＧＴ−ＡＧルール）に矛盾しないよう手動で再配置した。その結果配列番号：１の全ての領域で合理的な一致が得られた。
【０１３２】
【表２】

【０１３３】
Ｃ−ＴＥＳＴＩ２０５３６６７のクロモゾーム上の位置を特定するため配列番号：１をクエリーとしてヒトゲノム配列データベース（Ａｐｒ．１４，２００３）に対してＢＬＡＳＴ検索を実施した。得られた結果を通常期待されるエクソン−イントロン境界（ＡＧ−ＧＴルール）に矛盾しないよう手動で再配置した。その結果配列番号：３の全ての領域で合理的な一致が得られた。
また、転写開始点周辺のＴＡＴＡシグナル、イニシエーター（ＩＮＲ）シグナルの位置を図４および図５に示す。
実施例１から４の結果より、本発明遺伝子はヒトの完全長蛋白質をコードしている事が示された。
実施例５
（１）「Ｃ−ＢＲＡＣＥ２０４７５７３」の発現頻度解析ははその塩基配列に含まれる新規性のある領域に特異的なプライマーセット５’（ｔｇｃｔｃｃｔｇｃｔ ｇｃｔｃｔｔｔｇｃｔ／配列番号：９）と３’（ｃｃｔｃｃｔｔａｇａ ｃａｇａａｇｃｃｃｃ ａｃａ／配列番号：１０）を用い、クローンテック社のＭｕｌｔｉｐｌｅ Ｔｉｓｓｕｅ ｃＤＮＡ （ＭＴＣ（ＴＭ）） Ｐａｎｅｌｓ Ｈｕｍａｎ ＩおよびＨｕｍａｎ ＩＩの合計１６組織由来ｃＤＮＡ（心臓、脳、胎盤、肺、肝臓、筋肉、腎臓、膵臓、脾臓、胸腺、前立腺、精巣、卵巣、小腸、大腸および末梢白血球）をＰＣＲ法により増幅する事により行った。ＤＮＡ合成酵素としてポリメラーゼ（Ｔａｋａｒａ Ｔａｑ Ｐｏｌｙｍｅｒａｓｅ、Ｒ００１Ａ；タカラバイオ株式会社）を用い、鋳型ｃＤＮＡ ２．５ｎｇ、各プライマーを０．３μＭ、ｄＮＴＰを各０．１ｍＭを含む１倍希釈の酵素添付緩衝液となるよう調整した。各反応液は９２℃１分間の熱変性の後、９２℃１０秒、５８℃１０秒の温度サイクルを＜！−−標準化用遺伝子の、Ｃ−ＢＲＡＣＥ２０４７５７３およびＣ−ＴＥＳＴＩ２０５３６６７それぞれについて２５回、／／−−＞４３回＜！−−および３５回／／−−＞繰り返して増幅した。増幅産物は８％ アクリルアミドゲル電気泳動により分離し、臭化エチジウム染色後写真撮影することにより行った。
結果を図６に示す。
（２）「Ｃ−ＴＥＳＴＩ２０５３６６７」の発現頻度解析はその特異的プライマーセット５’（ｃａｔｔｔｇｃｔｇａ ｇｃｃｃｔｃｔｃｔｔ ｇｔ／配列番号：１１）と３’（ｇｃｔｇａｃｃｃｃａ ｃｔｔｔｃｔｃｔｔｇ ｇｔ／配列番号：１２）を用い、クローンテック社のＭｕｌｔｉｐｌｅ Ｔｉｓｓｕｅ ｃＤＮＡ （ＭＴＣ（ＴＭ）） Ｐａｎｅｌｓ Ｈｕｍａｎ ＩおよびＨｕｍａｎ ＩＩの合計１６組織由来ｃＤＮＡを鋳型として上記と同様な方法で行った。但し、温度サイクルを３５回繰り返した。
結果を図６に示した。
（３）コントロール遺伝子としてＧＡＰＤＨ（Ｇｌｙｃｅｒａｌｄｅｈｙｄｅ ３−Ｐｈｏｓｐｈａｔｅ Ｄｅｈｙｄｒｏｇｅｎａｓｅ）を上記同様に各鋳型ｃＤＮＡから増幅した。増幅にはＴａｑＭａｎ ＧＡＰＤＨ Ｃｏｎｔｒｏｌ Ｒｅａｇｅｎｔｓ（４０２８６９；アプライドバイオシステムズジャパン株式会社）に含まれるＧＡＰＤＨ Ｆｏｒｗａｒｄ ＰｒｉｍｅｒとＧＡＰＤＨ Ｒｅｖｅｒｓｅ Ｐｒｉｍｅｒを用い、上記と同様の条件で増幅した。但し、温度サイクルは２５回とした。
結果を図６に示した。
【０１３４】
【配列表】

【０１３５】
【産業上の利用の可能性】
本発明により、新規なヒトプロテインキナーゼ蛋白質、および該蛋白質をコードする遺伝子が提供された。キナーゼによる蛋白質のリン酸化状態の調節は、細胞の正常な分化・増殖、および細胞レベルでの生理機能にとって中心的な役割を担っている。本発明の新規キナーゼ・フォスファターゼも細胞内生理機能に深く関わっているものと考えられることから、本発明の蛋白質は、医薬品開発の上で薬剤の標的分子として有用である。また、本発明の蛋白質に作用する薬剤は、従来のレセプターアゴニスト・アンタゴニストに代表される薬剤よりも、より緻密に細胞内生理機能を調節し得る有効な医薬品となることが期待される。
特に、Ｃ−ＢＲＡＣＥ２０４７５７３は胎盤において強い発現が見られ、従って発生期において、特に本クローンの由来から小脳の発生期において重要な役割を果たしている新規スプライス変異体である事が示唆された。これにより、特に発生期の異常の診断、治療にも特に有効な利用が期待される。
また、Ｃ−ＴＥＳＴＩ２０５３６６７に関しては、白血球、脾臓、胸腺の様に免疫系組織に特に強く見出され、精巣、胎盤のような発生関連組織、および膵臓、肺に若干の発現が見られた。従って、この遺伝子では発生期の異常の診断、治療および免疫疾患の診断、治療において特に有益な利用が期待される。
【図面の簡単な説明】
【図１】実施例１で作成したベクターｐＭＥ１８ＳＦＬ３を示す。
【図２】ＣＬＵＳＴＡＬＷによるＣ−ＢＲＡＣＥ２０３７５７３とその相同性のトップヒット遺伝子とのアラインメントを示す。相同性のトップヒット遺伝子は、配列番号：２のペプチド配列をクエリーとして各データベース〔ＧｅｎＰｅｐｔ（ＮＣＢＩ）、ｒｅｆｓｅｑ（ＮＣＢＩ）及びＡａｇｅｎｅｓｅｑ（ダウエント社）〕に対するＢＬＡＳＴ検索を実施して得た。図中ｇｉ｜１０８６４６５０｜ｒｅｆはｒｅｆｓｅｑより得られたＰＫＣζ遺伝子の配列である。
【図３】ＣＬＵＳＴＡＬＷによるＣ−ＴＥＳＴＩ２０５３６６７とその相同性のトップヒット遺伝子とのアラインメントを示す。相同性のトップヒット遺伝子は、配列番号：２のペプチド配列をクエリーとして各データベース〔ＧｅｎＰｅｐｔ（ＮＣＢＩ）、ｒｅｆｓｅｑ（ＮＣＢＩ）及びＡａｇｅｎｅｓｅｑ（ダウエント社）〕に対するＢＬＡＳＴ検索を実施して得た。図中ｇｉ｜１０７２７２６４｜ｇｂはＧｅｎＰｅｐｔより得られた機能未知の遺伝子の配列である。
【図４】表３の結果の第一エキソンの上流にある転写開始シグナルを検討した。図５と共に、ゲノム中 ｕｎｐｌａｃｅｄ＃ｃｏｎｔｉｇｓ．ｆａ中ＮＴ＿０７７９８４．１の ２７００１から２７８２０の領域を示す。図中配列の上部の数字は２７００１から数えた塩基の位置を示す。イニシエーター配列コンセンサス配列（Ｃ／Ｔ Ｃ／Ｔ Ａ Ｎ Ｔ／Ａ Ｃ／Ｔ Ｃ／Ｔ）および、その上流−４５から−２５の位置にあるＡＴリッチ領域の存在から予測された転写開始点は配列番号：１の５’末端と一致した。
【図５】表３の結果の第一エキソンの上流にある転写開始シグナルを検討した。図４と共に、ゲノム中 ｕｎｐｌａｃｅｄ＃ｃｏｎｔｉｇｓ．ｆａ中ＮＴ＿０７７９８４．１の ２７００１から２７８２０の領域を示す。図中配列の上部の数字は２７００１から数えた塩基の位置を示す。イニシエーター配列コンセンサス配列（Ｃ／Ｔ Ｃ／Ｔ Ａ Ｎ Ｔ／Ａ Ｃ／Ｔ Ｃ／Ｔ）および、その上流−４５から−２５の位置にあるＡＴリッチ領域の存在から予測された転写開始点は配列番号：１の５’末端と一致した。
【図６】表３の結果の第一エキソンの上流にある転写開始シグナルを検討した。図７と共に、ゲノム中 ｃｈｒ１３．ｆａ中ＮＣ＿００００１３．５の ２７７６０００１から２７７６０６８０の領域を示す。図中配列の上部の数字は２７７６０００１から数えた塩基の位置を示す。イニシエーター配列コンセンサス（Ｃ／Ｔ Ｃ／Ｔ Ａ Ｎ Ｔ／Ａ Ｃ／Ｔ Ｃ／Ｔ）およびその上流−３１の位置にあるＡＴリッチ領域の存在から予測された転写開始点は配列番号：１の５’末端と一致した。
【図７】表３の結果の第一エキソンの上流にある転写開始シグナルを検討した。図６と共に、ゲノム中 ｃｈｒ１３．ｆａ中ＮＣ＿００００１３．５の ２７７６０００１から２７７６０６８０の領域を示す。図中配列の上部の数字は２７７６０００１から数えた塩基の位置を示す。イニシエーター配列コンセンサス（Ｃ／Ｔ Ｃ／Ｔ Ａ Ｎ Ｔ／Ａ Ｃ／Ｔ Ｃ／Ｔ）およびその上流−３１の位置にあるＡＴリッチ領域の存在から予測された転写開始点は配列番号：１の５’末端と一致した。
【図８】本発明遺伝子の発現頻度解析の結果をＰＣＲ増幅産物の泳動像として示す。Ｃ−ＢＲＡＣＥ２０４７５７３は胎盤において強い発現が見られた。また、Ｃ−ＴＥＳＴＩ２０５３６６７に関しては、白血球、脾臓、胸腺の様に免疫系組織に特に強く見出され、精巣、胎盤のような発生関連組織、および膵臓、肺に若干の発現が見られた。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to genes encoding novel protein kinases and protein kinase splice variants.
[0002]
[Prior art]
In order for cells to maintain homeostasis, to differentiate and proliferate normally as needed, and to exert functions at the tissue level, the various physiological functions of cells must be controlled correctly and in harmony. I must. It is well known that protein phosphorylation plays a central role in many of such control functions.
[0003]
Eukaryotic phosphorylation is carried out by transferring a high-energy phosphate group at the γ-position of ATP to a specific residue of a specific protein by protein kinase (hereinafter referred to as protein kinase). However, to date, a large number of protein kinase genes have been identified, and it has been shown that they constitute a very large family of proteins that are structurally well conserved.
[0004]
[Non-Patent Document 1]
Hanks
FASEB J 1995 May; 9: 576-96
[0005]
[Non-Patent Document 2]
Manning Science 2003 Dec; 298: 1912-34 The phosphorylation reaction is a very important reaction in controlling in vivo reactions because it changes the three-dimensional structure of the molecule and changes its properties instantaneously. Protein phosphorylation occurs due to extracellular signals such as hormones, neurotransmitters, growth and differentiation factors, cell cycle checkpoints, environmental and nutritional stress. Examples of proteins that are activated by phosphorylation include metabolic enzymes, regulatory proteins, receptors, cytoskeletal proteins, ion channels or ion pumps, and transcription factors.
Thus, protein kinases control the growth, differentiation, and signal transduction of various cells by transferring phosphate groups to proteins.
[0006]
[Non-Patent Document 3]
Hunter T. , Cell 1987 Sep 11; 50 (6): 823-9.
This abnormal signaling has also been shown to cause diseases such as inflammation, cancer, arteriosclerosis, and psoriasis.
[0007]
[Non-Patent Document 4]
Hunter T. Harvey Lect 1998-99; 94: 81-119.
[0008]
The various physiological reactions as described above are finely controlled by protein kinases present in large numbers in the cells. Therefore, drugs acting on protein kinases are more precise than existing drugs typified by receptor agonists and receptor antagonists. It is thought that there is a possibility that physiological functions can be suppressed. Protein kinase agonists are expected to be highly beneficial drugs that can dissociate undesirable side effects from their main effects.
[0009]
It is expected that many protein kinases have been isolated and studied to date, but many have not yet been identified. In addition, for the genes that have been isolated, knowledge about the intracellular physiological functions involved with each protein kinase is still very poor, and most of them have not yet been elucidated. By identifying new protein kinases and elucidating their physiological functions, it is expected that significant progress will be made in the development of new drugs and therapeutics.
[0010]
In addition, many protein kinases produce peptides with different amino acid sequences in certain regions by changing exon regions and / or combinations during the maturation process after transcription (see below). Called splice variant). Many protein kinases are known that change substrate specificity for each splice variant and are thought to play different roles in intracellular signal transduction pathways [for example, MKK3 (Enslen et al. The EMBO Journal 2000 Mar; 19 (6): 1301-11), IRAK (J. Biol. Chem 2001; 276 (31): 29037-44), p38α (Biochem. Biophys. Res. Comm. 2002; 291: 838-43), DCLK (J. Biol. Chem. 2002; 277 (20): 17696-705), PDK-1β (Biochem. Biophys. Res. Comm.), CK1α (J. Cell Biochem. 2002; 6 (4): 805 over 14), JNK (Biochem.J 2003; 370:. 159-66), STE20 (Mol.Cell.Biol 2003 Mar; 23 (6.): 2068-82)]. As shown in many of these examples, protein kinases are not only controlled gene by gene but also for each mutant in maintaining cellular function and homeostasis, and discovery of new mutants and physiological functions Analysis is expected to bring about significant progress in understanding disease states, developing new medicines and developing treatments.
[0011]
[Problems to be solved by the invention]
As described above, the present inventors have made various searches for new proteins belonging to protein kinases having great utility. As a result, the human cerebellum and
Succeeded in identifying a novel protein kinase gene and a novel protein kinase splice variant from a human testis-derived cDNA library,
The present invention has been completed.
[0012]
The present invention relates to a novel protein kinase protein, a novel protein kinase splice variant, and a gene encoding the protein,
In addition, it is an object to provide a manufacturing method and use thereof.
[0013]
DISCLOSURE OF THE INVENTION
The present invention has been isolated by the Helix Laboratory,
A clone whose structure has been determined (hereinafter referred to as a helix clone; Japanese Patent Application No. 11-248036, Japanese Patent Application No. 2000-118776,
This can be achieved by selecting a clone having a protein kinase-like structure from Japanese Patent Application No. 2000-183767). This helix clone
[1] Preparation of a cDNA library having a high full length ratio by the oligo cap method and
[2] Evaluation system for full-length property from 5 'terminal sequence (excluding non-full-length sequences from EST, then clustered to select representative sequence)
It is a clone with a high probability of being a full length obtained by the combination.
In addition, since cDNA is incorporated into an expression vector for mammalian cells, it has an advantage that expression experiments in cells can be performed immediately.
[0014]
Therefore, the inventors performed a hidden Markov model search of the known protein kinase catalytic domain for all of the helix clones,
Furthermore, two clones “C-BRACE2047573” and “C-TESTI2053667” were selected from the examination on the novelty of the sequence. These include cDNAs encoding novel human proteins and novel splice variants of known protein kinases.
Many known protein kinases are known to be involved in various intracellular signal transduction pathways, and the clones with the protein kinase-like structure found this time are also involved in some signal transduction pathways. There is a possibility.
By evaluating these in an assay system using a reporter gene or an experiment using RNAi, it is possible to analogize the physiological function and explore potential as a drug discovery target molecule. It is done.
[0015]
As described above, the present inventors have developed a novel protein kinase protein, a novel splice variant, and a gene encoding the protein,
In addition, regarding their manufacture and applications, more specifically
(A) The DNA according to any one of (a) to (d) below.
(A) A DNA encoding a protein containing a novel region from No. 1 to No. 189 in the amino acid sequence shown in Sequence Listing 2, or a DNA encoding a novel protein of Sequence Listing 4.
[0016]
(B) DNA containing a first exon region from No. 1 to No. 668 shown in Sequence Listing 1 or a novel DNA containing a coding region shown in Sequence Listing 3.
(C) one or more amino acids in the amino acid sequence of the novel region in Sequence Listing 2 or Sequence Listing 4 have an amino acid sequence substituted, deleted, inserted and / or added; DNA encoding a functionally equivalent protein.
[0017]
(D) DNA having a novel region in Sequence Listing 1 or Sequence Listing 3 and stringent conditions
A DNA encoding a protein functionally equivalent to a protein consisting of the amino acid sequence set forth in Sequence Listing 2 or Sequence Listing 4
[0018]
(B) DNA encoding a protein and a partial peptide having the amino acid sequence described in Sequence Listing 2 or Sequence Listing 4 described in A.
(C) A protein or peptide encoded by the DNA described in A or B.
(D) A vector into which the DNA described in A or B is inserted
(E) A host cell carrying the DNA described in A or B or the vector described in D.
(F) culturing the host cell according to E, and recovering the expressed protein from the host cell or its culture supernatant,
A method for producing the protein or peptide thereof according to C.
(G) An antibody that binds to the protein according to C.
(H) A polynucleotide comprising at least 15 nucleotides complementary to DNA consisting of the novel region described in Sequence Listing 1 or Sequence Listing 3 described in A or a complementary strand thereof.
(I) A screening method for a compound that binds to the protein according to C,
(A) contacting the test sample with the protein or its partial peptide, (b) detecting the binding activity between the protein or its partial peptide and the test sample,
(C) selecting a compound having an activity of binding to the protein or a partial peptide thereof,
Including methods.
Is to provide.
[0019]
The present invention provides a human-derived gene “C-BRACE2047573” encoding a novel protein kinase splice variant and a human-derived gene “C-TESTI2053667” encoding a novel protein kinase. The nucleotide sequence of the human-derived gene “C-BRACE2047573” cDNA is shown in Sequence Listing 1, and the amino acid sequence of the protein encoded by the cDNA is shown in Sequence Listing 2. The nucleotide sequence of the human-derived gene “C-TESTI2053667” cDNA is shown in Sequence Listing 3, and the amino acid sequence of the protein encoded by the cDNA is shown in Sequence Listing 4. “C-BRACE2047573” shown in Sequence Listing 1 has an ORF encoding a protein consisting of 457 amino acids, and “C-TESTI2053667” shown in Sequence Listing 3 has an ORF encoding a protein consisting of 483 amino acids.
[0020]
The present invention also includes proteins functionally equivalent to the protein of the present invention (Sequence Listing 2 or Sequence Listing 4). Examples of such proteins include substitution mutant homologues of the protein of the present invention. In the present specification, the substitution mutant includes a mutant containing mutation, insertion and deletion of 1 to 50 amino acids, particularly 10 amino acids or less. Here, “functionally equivalent” means that the target protein has a function of phosphorylating the protein in the same manner as the protein of the present invention. Whether or not the target protein phosphorylates the protein can be determined by the following method.
[0021]
The protein kinase protein and the substrate protein are mixed in an appropriate reaction solution, reacted in the presence of ATP, and then the phosphorylation activity of the substrate protein is measured to determine the phosphorylation activity. The protein kinase protein can be used after being purified from an appropriate cell line or tissue extract by a general biochemical method. In addition, genes that express protein kinase protein in mammalian cells (COS7, CV-1, HEK293, HeLa, Jurkat, NIH3T3, etc.), insect cells (Sf9, etc.), Escherichia bacteria (E. coli), yeast, etc. It is also possible to use a protein kinase protein introduced and expressed in large quantities. By using ATP labeled with a radioisotope such as [γ-32P] ATP, the phosphorylation state of the substrate protein can be measured by a liquid scintillation counter, autoradiography or the like.
[0022]
In addition, the phosphorylation state of the substrate protein can be measured by ELISA (enzyme-linked immunosorbent assay) or Western blotting using a phosphorylated protein-specific antibody. As the substrate protein, a protein specific to a specific protein kinase can be used, and a protein such as casein, histone, or myelin basic protein (MBP) generally used as a substrate for a protein kinase can also be used. Alternatively, a synthetic peptide having a phosphorylated sequence can also be used.
[0023]
Furthermore, phosphorylation activity can also be determined by measuring phosphorylation (autophosphorylation) of protein kinase protein itself. More specifically, Protein Phosphorylation: A Practical Approach. It can be performed according to a general method described in a book such as First Edition (Hardie DG. Et al., Oxford University Press., 1993).
[0024]
As a method for preparing a protein functionally equivalent to a certain protein, a method for introducing a mutation into the protein is known. For example, site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol. 100, 468-500, Kramer. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) Proc Natl Acad Sci US. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-2766) and the like. By introducing an appropriate mutation in Amino Acids, it can be prepared protein functionally equivalent to the protein. Amino acid mutations can also occur in nature. Thus, a protein having an amino acid sequence in which one or more amino acids are mutated in the amino acid sequence of the protein of the present invention (SEQ ID NO: 2 or 4) and functionally equivalent to the protein is also included in the protein of the present invention. included. In such mutants, the number of amino acids to be mutated is usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (for example, within 5 amino acids).
[0025]
The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved.
For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acid having an aliphatic side chain (A, V, L, I, P), amino acid having a hydroxyl group-containing side chain (S, T, Y), having a sulfur atom-containing side chain Amino acids (C, M), Amino acids (D, N, E, Q) with carboxylic acid and amide-containing side chains, Amino acids (R, K, H) with base-containing side chains, Aromatic-containing side chains Examples include amino acids (H, F, Y, W).
[0026]
It is already known that proteins having amino acid sequences modified by deletion, addition and / or substitution by other amino acids of one or more amino acid residues to a certain amino acid sequence maintain their biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5562-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487-. 6500, Wang, A. et al., Science 224, 1431-1433, Dalbadee-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).
[0027]
The protein having a plurality of amino acid residues added to the amino acid sequence of the protein of the present invention includes a fusion protein containing the protein of the present invention. The fusion protein is a fusion of the protein of the present invention and another peptide or protein, and is included in the present invention. The fusion protein is prepared by ligating the DNA encoding the protein of the present invention (SEQ ID NO: 2 or 4) and the DNA encoding another peptide or protein so that their frames coincide with each other by a known method. May be introduced into an expression vector and expressed in a host, and the other peptide or protein subjected to fusion with the protein of the present invention is not particularly limited.
[0028]
Examples of other peptides subjected to fusion with the protein of the present invention include FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), (6 His (histidine)). 6 × His, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T Antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein
Known peptides such as C fragments can be used. Examples of other proteins attached to the fusion of the protein of the present invention include GST (glutathione-S-transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP (maltose binding). Protein). A fusion protein can be prepared by fusing a commercially available DNA encoding the peptide or protein with a DNA encoding the protein of the present invention and expressing the prepared fusion DNA.
[0029]
As another method for preparing a protein functionally equivalent to a certain protein, a hybridization technique (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. press, 1989). That is, based on a DNA sequence encoding the protein of the present invention (SEQ ID NO: 1 or 3) or a part thereof, DNA having high homology with this is isolated and functionally equivalent to the protein of the present invention from the DNA. It is also usually possible to isolate such proteins. The present invention includes proteins that are encoded by a DNA that hybridizes with the DNA encoding the protein of the present invention and functionally equivalent to the protein of the present invention. Examples of such proteins include human and other mammalian homologs (eg, proteins encoded by mice, rats, rabbits, cows, etc.).
[0030]
Examples of hybridization conditions for isolating a DNA encoding a protein functionally equivalent to the protein of the present invention include low stringency conditions. The low stringent conditions are, for example, 42 ° C., 2 × SSC, 0.1% SDS, and preferably 50 ° C., 2 × SSC, 0.1% SDS in washing after hybridization. is there. More preferable hybridization conditions include highly stringent conditions. Highly stringent conditions are, for example, conditions of 65 ° C., 0.1 × SSC and 0.1% SDS. Under these conditions, it can be expected that DNA having high homology can be efficiently obtained as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and the same stringency can be realized by appropriately selecting these factors. Further guidance regarding hybridization conditions can be found in, for example, Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY), and Ausubel et al. (1995, Current in Protocol). Biology, John Wiley & Sons, NY).
[0031]
Further, in place of hybridization, a gene amplification method using primers synthesized based on the sequence information of the DNA encoding the protein of the present invention (SEQ ID NO: 1 or 3), for example, polymerase chain reaction (PCR) method is used. It is also possible to isolate them.
[0032]
Proteins functionally equivalent to the protein of the present invention encoded by DNA isolated by these hybridization techniques and gene amplification techniques usually have high amino acid sequence homology with the protein of the present invention (Sequence Listing 2 or 4). Have The protein of the present invention also includes proteins that are functionally equivalent to the protein of the present invention and have high homology with the amino acid sequences shown in Sequence Listing 2 or Sequence Listing 4. High homology generally refers to at least 65% identity, preferably 75% identity, more preferably 85% identity, most preferably 95% identity at the amino acid level. . To determine protein homology, the algorithm described in the literature (Wilbur, WJ and Lipman, DJ Proc. Natl. Acad. Sci. USA (1983) 80, 726-730) was used. Just do it.
[0033]
The protein of the present invention may differ in amino acid sequence, molecular weight, isoelectric point, presence / absence of sugar chain, form, etc., depending on the cell, host or purification method producing the protein, which will be described later. However, as long as the obtained protein has a function equivalent to the protein of the present invention, it is included in the present invention. For example, when the protein of the present invention is expressed in prokaryotic cells such as Escherichia, a methionine residue is added to the N-terminus of the amino acid sequence of the original protein. Such a protein is also included in the protein of the present invention.
[0034]
The protein of the present invention can be prepared as a recombinant protein or a natural protein by a known method. If it is a recombinant protein, a DNA encoding the protein of the present invention (for example, a DNA having the base sequence described in Sequence Listing 1 or Sequence Listing 3) is incorporated into an appropriate expression vector and introduced into an appropriate host cell. The obtained transformant is recovered and an extract is obtained, followed by chromatography such as ion exchange, reverse phase, gel filtration, or affinity chromatography in which an antibody against the protein of the present invention is immobilized on a column, Alternatively, it can be purified and prepared by combining a plurality of these columns.
[0035]
When the protein of the present invention is expressed in a host cell (for example, animal cell or Escherichia) as a fusion protein with glutathione S-transferase protein or as a recombinant protein to which a plurality of histidines are added, The expressed recombinant protein can be purified using a glutathione column or a nickel column. After purification of the fusion protein, the region other than the target protein in the fusion protein can be cleaved and removed with thrombin or factor Xa as necessary.
[0036]
In the case of a natural protein, for example, by purifying an extract of tissue or cells expressing the protein of the present invention by applying an affinity column bound with an antibody that binds to the protein of the present invention described later. It can be isolated. The antibody may be a polyclonal antibody or a monoclonal antibody.
[0037]
The present invention also includes partial peptides of the protein of the present invention. The partial peptide of the present invention consists of an amino acid sequence of at least 7 amino acids or more, preferably 8 amino acids or more, more preferably 9 amino acids or more. The partial peptide can be used, for example, for preparing an antibody against the protein of the present invention, screening for a compound that binds to the protein of the present invention, and screening for a promoter or inhibitor of the protein of the present invention. Further, it can be an antagonist or competitive inhibitor of the protein of the present invention. The partial peptide of the present invention can be produced by genetic engineering techniques, known peptide synthesis methods, or by cleaving the protein of the present invention with an appropriate peptidase. The peptide may be synthesized by, for example, either a solid phase synthesis method or a liquid phase synthesis method.
[0038]
The DNA encoding the protein of the present invention can be used for in vivo and in vitro production of the protein of the present invention as described above, for example, diseases caused by abnormalities in the gene encoding the protein of the present invention or the present invention. It can also be applied to gene therapy for diseases that can be treated with other proteins. The DNA of the present invention may be in any form as long as it can encode the protein of the present invention. That is, it does not matter whether it is cDNA synthesized from mRNA, genomic DNA, or chemically synthesized DNA. Moreover, as long as the protein of the present invention can be encoded, DNA having an arbitrary base sequence based on the degeneracy of the genetic code is included.
[0039]
The DNA of the present invention can be prepared by a known method. For example, by preparing a cDNA library from cells expressing the protein of the present invention and performing hybridization using a part of the DNA sequence of the present invention (for example, Sequence Listing 1 or Sequence Listing 3) as a probe. Can be prepared. The cDNA library may be prepared, for example, by the method described in the literature (Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)), or a commercially available DNA library may be used. Good. In addition, after preparing RNA from cells expressing the protein of the present invention, synthesizing cDNA with reverse transcriptase, oligo DNA based on the sequence of the DNA of the present invention (for example, Sequence Listing 1 or Sequence Listing 3) Can be prepared by amplifying the cDNA encoding the protein of the present invention by performing a PCR reaction using this as a primer.
[0040]
Further, by determining the base sequence of the obtained cDNA, the translation region encoded by the cDNA can be determined, and the amino acid sequence of the protein of the present invention can be obtained. Further, genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.
[0041]
Specifically, it may be performed as follows. First, mRNA is isolated from cells, tissues, and organs that express the protein of the present invention. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC method (Chomczynski, P. and Sacchi, N.). , Anal.Biochem. (1987) 162, 156-159), etc., and mRNA is purified from the total RNA using mRNA Purification Kit (Pharmacia) or the like. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).
[0042]
CDNA is synthesized from the obtained mRNA using reverse transcriptase. The synthesis of cDNA can also be performed using AMV Reverse Transcrispase First-strand cDNA Synthesis Kit (Seikagaku Corporation). In addition, 5′-Ampli FINDER RACE Kit (manufactured by Clontech) and 5′-RACE method using PCR (Frohman, MA et al., Proc. Natl) using primers and the like described in the present specification. Acad.Sci.U.S.A. (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932). be able to.
[0043]
A target DNA fragment is prepared from the obtained PCR product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia, etc., and colonies are selected to prepare a desired recombinant vector. The base sequence of the target DNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.
[0044]
Further, in the DNA of the present invention, a base sequence with higher expression efficiency can be designed in consideration of the codon usage frequency of the host used for expression (Grantham, R. et al., Nucleic Acids Research (1981) ) 9, r43-74). The DNA of the present invention can be modified by a commercially available kit or a known method. Modifications include, for example, digestion with restriction enzymes, insertion of synthetic oligonucleotides and appropriate DNA fragments, addition of linkers, insertion of start codon (ATG) and / or stop codon (TAA, TGA, or TAG). .
[0045]
Specifically, the DNA of the present invention includes DNA consisting of base A at position 102 to base G at position 1472 in the base sequence of Sequence Listing 1. In addition, it includes DNA consisting of base A at position 36 to G at position 1451 in the base sequence of Sequence Listing 3.
[0046]
The DNA of the present invention is also a DNA that hybridizes with a novel region of Sequence Listing 1 or a DNA comprising the base sequence shown in Sequence Listing 3, and encodes a protein that is functionally equivalent to the protein of the present invention. Containing DNA. The conditions for hybridization can be appropriately selected. Specifically, the above-described conditions can be used. Under these conditions, DNA having high homology can be obtained as the temperature is increased. The hybridizing DNA is preferably a naturally occurring DNA, such as cDNA or chromosomal DNA.
[0047]
The present invention also provides a vector into which the DNA of the present invention has been inserted.
The vector of the present invention is useful for retaining the DNA of the present invention in a host cell or expressing the protein of the present invention.
[0048]
As a vector, for example, when Escherichia is used as a host, the vector is amplified in Escherichia in order to amplify it in large quantities with Escherichia (for example, JM109, DH5α, HB101, XL1Blue), etc. In addition, there must be a selection gene for transformed Escherichia bacteria (for example, a drug resistance gene that can be distinguished by any drug (ampicillin, tetracycline, kanamycin, chloramphenicol)). There are no particular restrictions. Examples of vectors include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script and the like. In addition, for the purpose of subcloning and excision of cDNA, in addition to the above vectors, for example, pGEM-T, pDIRECT, pT7 and the like can be mentioned.
[0049]
An expression vector is particularly useful when a vector is used for the purpose of producing the protein of the present invention. As an expression vector, for example, for the purpose of expression in Escherichia genus, in addition to the above characteristics that the vector is amplified in Escherichia genus, the host is JM109, DH5α, HB101, XL1-Blue, etc. In the genus Escherichia, such as lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422- 2427), the araB promoter (Better et al., Science (1988) 240, 1041-1043), or the T7 promoter is essential. Examples of such a vector include pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase). BL21 is preferred).
[0050]
The vector may contain a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, the pelB signal sequence (Lei, SP et al J. Bacteriol. (1987) 169, 4379) may be used in the case of producing it in the periplasm of the genus Escherichia. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.
[0051]
In addition to the genus Escherichia, for example, as a vector for producing the protein of the present invention, an expression vector derived from a mammal (for example, pcDNA3 (manufactured by Invitrogen)) or pEGF-BOS (Nucleic Acids. Res. 1990). , 18 (17), p5322), pEF, pCDM8), insect cell-derived expression vectors (eg, “Bac-to-BAC baculovirus expression system” (manufactured by Invitrogen), pBacPAK8), plant-derived expression vectors (eg, pMH1) PMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “Pichia Xpression Kit "(Invitrogen), pNV11, SP-Q01), expression vectors derived from Bacillus subtilis (eg, pPL608, pKTH50) can be mentioned.
[0052]
For the purpose of expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, promoters required for expression in cells such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have an MMLV-LTR promoter, an EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), a CMV promoter, etc., and a gene for selecting transformation into cells (for example, It is more preferable if it has a drug (drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.)). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
[0053]
Furthermore, when the gene is stably expressed and the purpose is to amplify the copy number of the gene in the cell, a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway (for example, , PCHOI, etc.) and amplifying with methotrexate (MTX). For the purpose of transient expression of the gene, COS having a gene expressing SV40 T antigen on the chromosome Examples thereof include a method of transforming with a vector (such as pcD) having an SV40 replication origin using cells. As the replication origin, those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used. Furthermore, in order to amplify the gene copy number in the host cell system, the expression vector can be used as a selection marker such as aminoglycoside transferase (APH) gene, thymidine protein kinase (TK) gene, Escherichia genus xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate. A reductase (dhfr) gene can be included.
[0054]
On the other hand, as a method for expressing the DNA of the present invention in the living body of an animal, the DNA of the present invention is incorporated into an appropriate vector. The method introduced into Thereby, it is possible to perform gene therapy for a disease caused by the mutation of the gene of the present invention. Examples of the vector to be used include, but are not limited to, an adenovirus vector (for example, pAdexlcw) and a retrovirus vector (for example, pZIPneo). General genetic manipulation such as insertion of the DNA of the present invention into a vector can be performed according to a conventional method (Molecular Cloning, 5.61-5.63). Administration to a living body may be an ex vivo method or an in vivo method.
[0055]
The present invention also provides a host cell into which the vector of the present invention has been introduced. The host cell into which the vector of the present invention is introduced is not particularly limited, and for example, Escherichia bacteria and various animal cells can be used. The host cell of the present invention can be used, for example, as a production system for production and expression of the protein of the present invention. Production systems for protein production include in vitro and in vivo production systems. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.
[0056]
When eukaryotic cells are used, for example, animal cells, plant cells, and fungal cells can be used as the host. Animal cells include mammalian cells such as CHO (J. Exp. Med. (1995) 108,945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian cells, such as Xenopus laevis. Oocytes (Vale, et al., Nature (1981) 291, 358-340) or insect cells such as Sf9, Sf21, Tn5 are known. Examples of CHO cells include dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO K-1 (Proc. Natl. Acad. Cadmium), which are CHO cells deficient in the DHFR gene. Sci. USA (1968) 60, 1275) can be preferably used. In animal cells, CHO cells are particularly preferred for the purpose of mass expression. Introduction of the vector into the host cell can be performed by, for example, a calcium phosphate method, a DEAE dextran method, a method using a cationic ribosome DOTAP (Boehringer Mannheim), an electroporation method, a lipofection method, or the like.
[0057]
As plant cells, for example, cells derived from Nicotiana tabacum are known as protein production systems, and these may be cultured in callus. Fungal cells include yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, filamentous fungi such as the genus Aspergillus, such as Aspergillus niger sulgi. .
[0058]
When prokaryotic cells are used, there are production systems using bacterial cells. Examples of bacterial cells include Escherichia genus (E. coli), such as JM109, DH5α, HB101, and others, and Bacillus subtilis is known.
[0059]
Proteins are obtained by transforming these cells with the target DNA and culturing the transformed cells in vitro. The culture can be performed according to a known method. For example, DMEM, MEM, RPMI1640, and IMDM can be used as the culture medium for animal cells. At that time, a serum supplement such as fetal calf serum (FCS) can be used in combination, or serum-free culture can be performed. The pH during the culture is preferably about 6-8. The culture is usually performed at about 30 to 40 ° C. for about 15 to 200 hours, and medium exchange, aeration, and agitation are added as necessary.
[0060]
On the other hand, examples of a system for producing a protein in vivo include a production system using animals and a production system using plants. A target DNA is introduced into these animals or plants, and proteins are produced and collected in the animals or plants. The “host” in the present invention includes these animals and plants.
[0061]
When animals are used, there are production systems using mammals. As mammals, goats, pigs, sheep, mice, and cattle can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Moreover, when using a mammal, a transgenic animal can be used.
[0062]
For example, the target DNA is prepared as a fusion gene with a gene encoding a protein inherently produced in milk such as goat β casein. Next, a DNA fragment containing the fusion gene is injected into a goat embryo, and the embryo is transferred to a female goat. The protein of interest can be obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its progeny. Hormones may be used as appropriate in transgenic goats to increase the amount of milk containing protein produced from the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12,699. -702).
[0063]
Insects can also be used in the above. For example, when silkworms are used, by infecting silkworms with baculovirus inserted with DNA encoding the target protein,
The desired protein can be obtained from the body fluid of this silkworm (Susumu, M. et al., Nature (1985) 315, 592-594).
[0064]
Furthermore, when using plants, for example, tobacco can be used.
When using tobacco, a DNA encoding the protein of interest is inserted into a plant expression vector, such as pMON 530,
This vector is introduced into bacteria such as Agrobacterium tumefaciens.
Infecting this bacterium with tobacco, for example Nicotiana tabacum,
The desired polypeptide can be obtained from the leaves of this tobacco (Julian K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).
[0065]
The protein of the present invention thus obtained can be isolated from the inside of the host cell or outside the cell (such as a medium) and purified as a substantially pure and uniform protein. Separation and purification of proteins may be carried out using separation and purification methods used in usual protein purification, and are not limited in any way.
For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, immunoprecipitation,
Proteins can be separated and purified by appropriately selecting and combining SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization and the like.
[0066]
Examples of chromatography include affinity chromatography, ion exchange chromatography,
Examples include hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography.
These chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC.
The present invention also includes highly purified proteins using these purification methods.
[0067]
The protein can be arbitrarily modified or the peptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after purification of the protein. Examples of protein modifying enzymes include trypsin, chymotrypsin, lysyl endopeptidase, protein protein kinase, glucosidase and the like.
[0068]
The present invention also provides an antibody that binds to the protein of the present invention.
The form of the antibody of the present invention is not particularly limited, and includes monoclonal antibodies as well as polyclonal antibodies.
Also included are antisera obtained by immunizing animals such as rabbits with the protein of the present invention, all classes of polyclonal and monoclonal antibodies, human antibodies and humanized antibodies by genetic recombination.
[0069]
The protein of the present invention used as a sensitizing antigen for antibody acquisition is not limited to the animal species from which it is derived, but mammals,
For example, a protein derived from human, mouse or rat is preferable, and a protein derived from human is particularly preferable.
Human-derived proteins can be obtained using the gene sequences or amino acid sequences disclosed in this specification.
[0070]
In the present invention, the protein used as the sensitizing antigen may be a complete protein,
It may be a partial peptide of a protein. Examples of the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein.
The “antibody” described in the present specification means an antibody that reacts with the full length or fragment of a protein.
[0071]
A gene encoding the protein of the present invention or a fragment thereof is inserted into a known expression vector system, the host cell described in this specification is transformed with the vector, and the target protein or fragment thereof is known from inside or outside the host cell. These can be used as sensitizing antigens. Alternatively, cells expressing the protein, lysates thereof, or chemically synthesized proteins of the present invention may be used as the sensitizing antigen.
The short peptide is preferably combined with a carrier protein such as keyhole limpet hemocyanin, bovine serum albumin, ovalbumin and the like as an antigen.
[0072]
The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of the compatibility with the parent cell used for cell fusion. Rabbit eyes, primate animals are used.
[0073]
Examples of rodent animals include mice, rats, and hamsters.
For example, a rabbit is used as the animal of the order of rabbit eyes. For example, monkeys are used as the primate animals.
As monkeys, monkeys (old world monkeys) with narrow nose, such as cynomolgus monkeys, rhesus monkeys, baboons, chimpanzees and the like are used.
[0074]
In order to immunize a mammal with a sensitizing antigen, a known method is performed.
As a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously.
Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline,
If necessary, a normal adjuvant, for example, Freund's complete adjuvant, is mixed with the suspension in an appropriate amount. After emulsification, the suspension is administered to a mammal. Furthermore, it is preferable to administer a sensitizing antigen mixed in an appropriate amount with Freund's incomplete adjuvant several times every 4 to 21 days.
In addition, an appropriate carrier can be used during immunization with the sensitizing antigen.
Immunization is carried out in this manner, and it is confirmed by a conventional method that the desired antibody level is increased in the serum.
[0075]
Here, in order to obtain a polyclonal antibody against the protein of the present invention, after confirming that the desired antibody level in the serum was increased,
Remove the blood of the mammal sensitized with the antigen. Serum is separated from this blood by a known method.
As a polyclonal antibody, a serum containing a polyclonal antibody may be used, and if necessary, a fraction containing a polyclonal antibody may be further isolated from this serum and used.
For example, using an affinity column coupled with the protein of the present invention, a fraction that recognizes only the protein of the present invention is obtained, and this fraction is further purified using a protein A or protein G column. Immunoglobulin G or M can be prepared.
[0076]
To obtain a monoclonal antibody, after confirming that the desired antibody level is increased in the serum of a mammal sensitized with the antigen,
Immune cells may be removed from the mammal and subjected to cell fusion. In this case, spleen cells are particularly preferable as immune cells used for cell fusion.
The other parent cell to be fused with the immunocyte is preferably a mammalian myeloma cell, more preferably a myeloma cell that has acquired the characteristics for selecting fused cells by a drug.
[0077]
The cell fusion between the immunocytes and myeloma cells is basically performed by a known method such as the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46). It can be done as well.
[0078]
Hybridomas obtained by cell fusion can be prepared using conventional selective culture media such as HAT culture media (hypoxanthine,
Culture medium containing aminopterin and thymidine).
Culturing with the HAT culture solution is carried out for a time sufficient for the cells other than the target hybridoma (non-fused cells) to die, usually several days to several weeks.
Subsequently, a normal limiting dilution method is performed to screen and clone hybridomas that produce the target antibody.
[0079]
In addition to immunizing non-human animals with antigens to obtain the above hybridomas, they were infected with human lymphocytes such as EB virus.
Human lymphocytes are sensitized in vitro with proteins, protein-expressing cells or lysates thereof, and the sensitized lymphocytes have human-derived permanent mitotic potential
A hybridoma that produces a desired human antibody having a binding activity to a protein can also be obtained by fusing with a myeloma cell such as U266 (Japanese Patent Laid-Open No. 63-17688).
[0080]
Next, the obtained hybridoma was transplanted into the mouse abdominal cavity, ascites was collected from the mouse, and the obtained monoclonal antibody was
For example, it can be prepared by purification using ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the protein of the present invention, or the like.
The antibody of the present invention can be used for the purification and detection of the protein of the present invention and can also be a candidate for the agonist or antagonist of the protein of the present invention.
It is also conceivable to apply this antibody to antibody therapy for diseases involving the protein of the present invention.
When the obtained antibody is used for the purpose of administering it to the human body (antibody treatment), a human antibody or a human-type antibody is preferable in order to reduce immunogenicity.
[0081]
For example, a transgenic animal having a repertoire of human antibody genes is immunized with an antigen protein, a protein-expressing cell, or a lysate thereof to obtain an antibody-producing cell, and a hybridoma obtained by fusing this with a myeloma cell is used for the protein. Human antibodies can be obtained (see International Publication Nos. WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735 and WO96-34096).
[0082]
In addition to producing antibodies using hybridomas, cells obtained by immortalizing immune cells such as sensitized lymphocytes that produce antibodies with an oncogene may be used.
[0083]
The monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a gene recombination technique (for example, Borrebaeck, CAK and Larick, JW, (See THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).
Recombinant antibodies clone DNA encoding them from immune cells such as hybridomas or sensitized lymphocytes that produce antibodies,
It is incorporated into an appropriate vector and introduced into a host for production.
The present invention encompasses this recombinant antibody.
[0084]
Further, the antibody of the present invention may be an antibody fragment or a modified antibody as long as it binds to the protein of the present invention.
For example, as an antibody fragment, Fab, F (ab ′) 2, Fv, or a single chain Fv (scFv) in which Fv of H chain and L chain are linked by an appropriate linker (Huston, J. S. et al.,). Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883).
Specifically, the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, and then an appropriate host cell. (Eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178,476). -496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663, Rousseaux, J. et al. Methods En . Ymol (1986) 121,663-669; Bird, R.E.and Walker, B.W., see Trends Biotechnol (1991) 9,132-137)..
[0085]
Antibodies modified with various molecules such as polyethylene glycol (PEG) can also be used as modified antibodies.
The “antibody” of the present invention includes these modified antibodies. In order to obtain such a modified antibody, it can be obtained by chemically modifying the obtained antibody.
These methods are already established in this field.
[0086]
Moreover, the antibody of the present invention is derived from a chimeric antibody or a non-human antibody-derived CDR (complementarity determining region) and a human antibody comprising a variable region derived from a non-human antibody and a constant region derived from a human antibody using a known technique. It can be obtained as a humanized antibody consisting of the FR (framework region) and constant region.
[0087]
The antibody obtained as described above can be purified to homogeneity.
Separation and purification of antibodies used in the present invention may be carried out using separation and purification methods used for ordinary proteins.
For example, chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis,
By appropriately selecting and combining SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc., antibodies can be separated and purified (see Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988). ) Is not limited to these.
The concentration of the antibody obtained above can be measured by measuring absorbance or ELISA.
[0088]
Examples of columns used for affinity chromatography include a protein A column and a protein G column.
For example, as a column using a protein A column, Hyper D, POROS, Sepharose F. F. (Manufactured by Pharmacia).
[0089]
As chromatography other than affinity chromatography, for example, ion exchange chromatography,
Hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (see Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshar et al. ).
These chromatography can be performed using liquid phase chromatography, such as HPLC and FPLC.
[0090]
In addition, as a method for measuring the antigen binding activity of the antibody of the present invention, for example, absorbance measurement, ELISA, EIA (enzyme immunoassay),
RIA (radioimmunoassay) or fluorescent antibody method can be used.
When using ELISA, the protein of the present invention is added to a plate on which the antibody of the present invention is immobilized, and then a sample containing the target antibody,
For example, a culture supernatant of antibody-producing cells or a purified antibody is added.
Add a secondary antibody that recognizes an antibody labeled with an enzyme, such as alkaline phosphatase, and incubate the plate;
Next, after washing, antigen binding activity can be evaluated by adding an enzyme substrate such as p-nitrophenyl phosphate and measuring the absorbance.
[0091]
As the protein, a protein fragment, for example, a fragment consisting of the C-terminal thereof may be used.
BIAcore (Pharmacia) can be used for the activity evaluation of the antibody of the present invention.
[0092]
By using these techniques, the antibody of the present invention is brought into contact with the sample expected to contain the protein of the present invention contained in the sample,
The method for detecting or measuring a protein of the present invention comprising detecting or measuring an immune complex between the antibody and the protein can be carried out.
Since the protein detection or measurement method of the present invention can specifically detect or measure the protein, it is useful for various experiments using the protein.
[0093]
The present invention also provides a polynucleotide comprising at least 15 nucleotides complementary to DNA encoding the protein of the present invention (the novel region of Sequence Listing 1 or Sequence Listing 3) or its complementary strand.
[0094]
Here, the “complementary strand” refers to the other strand with respect to one strand of a double-stranded nucleic acid comprising A: T (U in the case of RNA) and G: C base pairs.
In addition, “complementary” is not limited to a case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, still more preferably 95%. What is necessary is just to have the homology on the above base sequences.
The algorithm described in the present specification may be used as an algorithm for determining homology.
[0095]
Such nucleic acids include probes and primers used for detection and amplification of DNA encoding the protein of the present invention, probes and primers for detecting the expression of the DNA, and nucleotides for controlling the expression of the protein of the present invention. Alternatively, nucleotide derivatives (for example, antisense oligonucleotides and ribozymes, or DNA encoding them) are included.
Moreover, such a nucleic acid can also be utilized for production of a DNA chip.
[0096]
When used as a primer, the region on the 3 ′ side is complementary, and a restriction enzyme recognition sequence or a tag can be added to the 5 ′ side.
[0097]
Antisense oligonucleotides include, for example, antisense oligonucleotides that hybridize to a novel region of Sequence Listing 1 or any position in the base sequence of Sequence Listing 3.
This antisense oligonucleotide is preferably an antisense oligonucleotide to at least 15 or more consecutive nucleotides in the novel region of Sequence Listing 1 or the base sequence of Sequence Listing 3. More preferably, it is an antisense oligonucleotide in which at least 15 or more consecutive nucleotides contain a translation initiation codon.
[0098]
As antisense oligonucleotides, derivatives or modifications thereof can be used.
As a modification, for example, a lower alkyl phosphonate modification such as methyl phosphonate type or ethyl phosphonate type,
Examples include phosphorothioate modified products and phosphoramidate modified products.
[0099]
Antisense oligonucleotides are not only those in which nucleotides corresponding to nucleotides constituting a predetermined region of DNA or mRNA are all complementary sequences, but also DNA or mRNA and oligonucleotide are novel regions of Sequence Listing 1 or As long as it can specifically hybridize to the base sequence shown in Sequence Listing 3, one containing one or more nucleotide mismatches is also included.
[0100]
The antisense oligonucleotide derivative of the present invention acts on the cells producing the protein of the present invention and binds to the DNA or mRNA encoding the protein, thereby inhibiting its transcription or translation or promoting the degradation of mRNA. Thus, by suppressing the expression of the protein of the present invention, the effect of suppressing the action of the protein of the present invention is obtained.
[0101]
The protein of the present invention is useful for screening for compounds that bind to the protein. That is, the protein of the present invention is brought into contact with a test sample expected to contain a compound that binds to the protein, and a compound having an activity that binds to the protein of the present invention is selected. Used in methods of screening for compounds that bind to proteins.
[0102]
The protein of the present invention used for screening may be a recombinant protein or a naturally derived protein.
It may also be a partial peptide. Moreover, the form expressed on the cell surface or the form as a membrane fraction may be sufficient.
There are no particular limitations on the test sample, for example, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract,
Examples include purified or roughly purified proteins, peptides, non-peptidic compounds, synthetic low molecular compounds, natural compounds, and the like.
The protein of the present invention to be contacted with a test sample is, for example, as a purified protein, a soluble protein, or a form bound to a carrier,
As a fusion protein with another protein, it can be brought into contact with a test sample as a membrane fraction as a form expressed on a cell membrane.
[0103]
As a method for screening for a protein that binds to the protein of the present invention, for example, many known methods can be used. Such screening can be performed, for example, by immunoprecipitation. Specifically, it can be performed as follows.
The gene encoding the protein of the present invention is inserted into a vector for expressing a foreign gene such as pSV2neo, pcDNAI, or pCD8 to express the gene in animal cells. As a promoter used for expression, SV40 early promoter (Rigby In Williamson (ed.), Genetic Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)), EF-1 α promoter Gene (K. 1982). , P.217-223 (1990)), CAG promoter (Niwa et al. Gene 108, p.193-200 (1991)), RSV LTR promoter (Cullen Methods in Enzymology 152, p. 684-704 (1987)). SR α promoter (Takebe et al. Mol. Cell. Biol. 8, p.466 (1988)), CMV im media early promoter (Seed and Aruffo Proc. Natl. Acad. Sci. USA 84, p. 3365-3369 (1987)), SV40 late promoter (Gheysen and Fiers J. Mol. App. 38, App. (1982)), Adenovirus late promoter (Kaufman et al. Mol. Cell. Biol. 9, p. 946 (1989)), HSV TK promoter, and any other generally usable promoter may be used.
[0104]
In order to express a foreign gene by introducing a gene into an animal cell, electroporation (see Chu, G. et al. Nucl. Acid Res. 15, 1311-1326 (1987)), calcium phosphate method (Chen , C and Okayama, H. Mol. Cell. Biol. 7, 2745-2752 (1987)), DEAE dextran method (Lopata, MA et al. Nucl. Acids Res. 12, 5707-5717 (1984). Sussman, D. J. and Milman, G. Mol. Cell. Biol. 4, 1644-143 (1985)), Lipofectin method (Derijard, B. Cell 7, 1025-1037 (1994); Lamb, B .; T. et al Nature Genetics 5,22-30 (1993); Rabindran, S.K.et al.Science 259,230-234 (1993) refer) there is a method such as may be by any method.
[0105]
By introducing a recognition site (epitope) of a monoclonal antibody whose specificity is clear into the N-terminus or C-terminus of the protein of the present invention, the protein of the present invention is expressed as a fusion protein having the recognition site of the monoclonal antibody. be able to. A commercially available epitope-antibody system can be used (see Experimental Medicine 13, 85-90 (1995)). A vector capable of expressing a fusion protein with β-galactosidase, maltose binding protein, glutathione S-transferase, green fluorescent protein (GFP) and the like via a multicloning site is commercially available.
[0106]
In order to avoid changing the properties of the protein of the present invention as much as possible by using a fusion protein, a method for preparing a fusion protein by introducing only a small epitope consisting of several to a dozen amino acids has also been reported. Yes. For example, polyhistidine (His-tag), influenza agglutinin HA, human c-myc, FLAG, vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV) -Tag), E-tag (epitope on monoclonal phage), and other monoclonal antibodies that recognize the epitope can be used as an epitope-antibody system for screening for proteins that bind to the protein of the present invention (Experimental Medicine 13). 85-90 (1995)).
[0107]
In immunoprecipitation, an immune complex is formed by adding these antibodies to a cell lysate prepared using an appropriate surfactant. This immune complex consists of the protein of the present invention, a protein capable of binding to the protein, and an antibody. In addition to using an antibody against the above epitope, immunoprecipitation can also be performed using an antibody against the protein of the present invention. The antibody against the protein of the present invention can be obtained by, for example, introducing a gene encoding the protein of the present invention into an appropriate Escherichia bacterium expression vector, expressing it in Escherichia bacterium, purifying the expressed protein, It can be prepared by immunizing mice, rats, goats, chickens and the like. It can also be prepared by immunizing the above animal with the synthesized partial peptide of the protein of the present invention.
[0108]
For example, if the antibody is a mouse IgG antibody, the immune complex can be precipitated using Protein A Sepharose or Protein G Sepharose. Further, when the protein of the present invention is prepared as a fusion protein with an epitope such as GST, for example, a substance that specifically binds to these epitopes such as glutathione-Sepharose 4B is used to Similar to the case of using an antibody, an immune complex can be formed.
[0109]
For general methods of immunoprecipitation, for example, according to the method described in the literature (see Harlow, E. and Lane, D .: Antibodies, pp. 511-552, Cold Spring Harbor Laboratory publications, New York (1988)), Or it may be performed according to the above.
[0110]
SDS-PAGE is generally used for analysis of immunoprecipitated proteins, and by using a gel with an appropriate concentration, proteins that are bound by the molecular weight of the protein can be analyzed. At this time, in general, the protein bound to the protein of the present invention is difficult to detect by ordinary protein staining methods such as Coomassie staining and silver staining, so that 35S-methionine, which is a radioactive isotope, Detection sensitivity can be improved by culturing cells in a culture solution containing 35S-cysteine, labeling the protein in the cells, and detecting this. If the molecular weight of the protein is known, the protein of interest can be purified directly from SDS-polyacrylamide gel and its sequence can be determined.
[0111]
Moreover, as a method for isolating a protein that binds to the protein using the protein of the present invention, for example, the West Western blotting method (Skolnik, EY et al., Cell (1991) 65, 83-90). Reference). That is, a cDNA library using a phage vector (λgt11, ZAP, etc.) is prepared from cells, tissues, or organs (eg, liver or kidney) that are expected to express a protein that binds to the protein of the present invention. A protein expressing the protein bound to the protein of the present invention by reacting the protein of the present invention, which is expressed on LB-agarose and expressed on the filter, immobilized, purified and labeled with the filter of the present invention What is necessary is just to detect with a label | marker. Examples of the method for labeling the protein of the present invention include a method using the binding property of biotin and avidin, an antibody that specifically binds to the protein of the present invention or a peptide or polypeptide fused to the protein of the present invention (eg, GST). , A method using a radioisotope, a method using fluorescence, and the like.
[0112]
In addition, as another aspect of the screening method of the present invention, a two-hybrid system using cells (Fields, S., and Sternglanz, R., Trends. Genet. (1994) 10, 286-292, Dalton S, and Treisman R (1992) Characterization of SAP-1, aprotein recruited by serum response factor to the c-fos serum response element.Cell 68,597-612, "MATCHMAKER Two-Hybrid System", "Mammalian MATCHMAKER Two-Hybrid Assay Kit ", MATCHMAKER One-Hybr d System "(both manufactured by Clontech), method using the" HybriZAP Two-Hybrid Vector System "(Stratagene) reference) and the like. In the two-hybrid system, the protein of the present invention or a partial peptide thereof is fused with the SRF DNA binding region or GAL4 DNA binding region and expressed in yeast cells to express a protein that binds to the protein of the present invention. A cDNA library that is expressed in a form fused with the VP16 or GAL4 transcriptional activation region is prepared from cells expected to be introduced into the yeast cell, and the library-derived cDNA is detected from the detected positive clone. (If a protein that binds to the protein of the present invention is expressed in yeast cells, the reporter gene is activated by the binding of the two, and a positive clone can be confirmed). By introducing the isolated cDNA into an Escherichia bacterium and expressing it, a protein encoded by the cDNA can be obtained. This makes it possible to prepare a protein that binds to the protein of the present invention or a gene thereof. Examples of the reporter gene used in the 2-hybrid system include, but are not limited to, the HIS3 gene, Ade2 gene, LacZ gene, CAT gene, luciferase gene, PAI-1 (plasminogen activator inhibitor type 1) gene, and the like. Not. Screening by the two-hybrid method can be performed using yeast, mammalian cells, and the like.
[0113]
Screening for a compound that binds to the protein of the present invention can also be performed using affinity chromatography. For example, the test sample that is expected to express a protein that binds to the protein of the present invention is applied to the protein of the present invention immobilized on a carrier of an affinity column. Examples of the test sample in this case include cell extracts and cell lysates. After applying the test sample, the column can be washed to prepare a protein bound to the protein of the present invention.
[0114]
The obtained protein is analyzed for its amino acid sequence, an oligo DNA is synthesized based on the amino acid sequence, and a cDNA library is screened using the DNA as a probe, whereby a DNA encoding the protein can be obtained.
[0115]
In the present invention, a biosensor using the surface plasmon resonance phenomenon can also be used as a means for detecting or measuring the bound compound. The biosensor using the surface plasmon resonance phenomenon allows the interaction between the protein of the present invention and the test compound to be observed in real time as a surface plasmon resonance signal without using a trace amount of protein and labeling. (For example, BIAcore, manufactured by Pharmacia). Therefore, it is possible to evaluate the binding between the protein of the present invention and the test compound by using a biosensor such as BIAcore.
[0116]
Further, methods for isolating compounds (including agonists and antagonists) that bind not only to proteins but also to proteins of the present invention include, for example, synthetic compounds, natural product banks, or random phages that are immobilized on proteins of the present invention. A method for screening a molecule that binds to the protein of the present invention by allowing a peptide display library to act, or a screening method using high-throughput by combinatorial chemistry technology (Wrightton NC; Farrell FX; Chang R; Kashhap AK; Barbone FP; Mulcahy LS; Johnson DL; Barret RW; Jolliffe LK; Dower WJ., Small peptides as potential mimics of the proto. in hormone erythropoietin, Science (UNITED STATES) Jul 26 1996,273 p458-64, Verdine GL., The combinatorial chemistry of nature.Nature (ENGLAND) Nov 7 1996,384 p11-13, Hogan JC Jr., Directed combinatorial chemistry. Nature (ENGLAND) Nov 7 1996, 384 p17-9) is known.
[0117]
A compound that can be isolated by the screening of the present invention is a candidate for a drug for regulating the activity of the protein of the present invention, and exhibits a disease caused by abnormal expression or abnormal function of the protein of the present invention or the activity of the protein of the present invention. Application to the treatment of diseases that can be treated by controlling is conceivable. Substances in which a part of the structure of a compound that can be isolated using the screening method of the present invention is converted by addition, deletion and / or substitution are also included in the compound that binds to the protein of the present invention.
[0118]
Use of the protein of the present invention or a compound that can be isolated by the screening of the present invention as a medicine for humans and animals such as mice, rats, guinea pigs, rabbits, chickens, cats, dogs, sheep, pigs, cows, monkeys, baboons, chimpanzees In this case, in addition to directly administering the protein or the isolated compound itself to the patient, it is also possible to administer it by formulating it by a known pharmaceutical method. For example, tablets, capsules, elixirs, microcapsules as required, or aseptic solutions or suspensions with water or other pharmaceutically acceptable liquids It can be used parenterally in the form of injections. For example, a pharmacologically acceptable carrier or medium, specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to formulate by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredients in these preparations is such that an appropriate volume within the indicated range can be obtained.
[0119]
Additives that can be mixed into tablets and capsules include, for example, binders such as gelatin, corn starch, gum tragacanth and gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin and alginic acid Lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry are used. When the dispensing unit form is a capsule, the above material may further contain a liquid carrier such as fats and oils. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
[0120]
Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethyleneglycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.
[0121]
Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.
[0122]
For example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, transdermal, or oral administration can be used as appropriate for administration to patients. The dose varies depending on the weight and age of the patient, the administration method, etc., but an appropriate dose can be appropriately selected according to the symptoms. In addition, if the compound can be encoded by DNA, it may be possible to incorporate the DNA into a gene therapy vector and perform gene therapy. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but an appropriate dose can be appropriately selected and used.
[0123]
【Example】
EXAMPLES Next, although an Example demonstrates this invention further more concretely, this invention is not limited to the following Example.
Example 1
Preparation of cDNA library by oligo cap method
(1) Preparation of cDNA library
MRNA extracted as total RNA from human tissue was purchased. The relationship between the library name and its origin is shown below in the order of “library name: origin, supplier”.
[0124]
BRACE: Brain, cerebellum, Clontech (CLONTECH # 64035-1)
TESTI: Oligocap method [M.C. from the testis (Testis) and Clontech (CLONTECH # 64027-1) RNAs. Maruyama and S.M. Sugano, Gene, 138: 171-174 (1994)] was used to prepare a cDNA library (International Patent Publication WO 01/04286). An oligo cap linker (Oligo-cap linker, SEQ ID NO: 5) and an oligo (dT) primer (Oligo dT primer, SEQ ID NO: 6) were used to treat BAP (Bacterial Alkaline Phosphatase), TAP as described in the publication. (Tobacco Acid Pyrophosphatase) treatment, RNA ligation, first-strand cDNA synthesis and RNA removal were performed. Next, it was converted into double-stranded cDNA by PCR (polymerase chain reaction) using 5 ′ (SEQ ID NO: 7) and 3 ′ (SEQ ID NO: 8) PCR primers, and cleaved with SfiI. Subsequently, the cDNA fragment was usually cloned into a vector pME18SFL3 (see FIG. 1) (GenBank AB009864, Expression vector) obtained by cleaving a cDNA fragment fractionated to 2 kb or more with DraIII to prepare a cDNA library.
(2) Evaluation of full length of 5′-end of clone from cDNA library prepared by oligocap method
Using the DNA sequencing reagent [BigDye Terminator Cycle Sequencing FS Ready Reaction Kit, manufactured by Bio Systems (PE Biosystems)], the sequencing reaction was performed on the plasmid DNA of the clones obtained from these using the DNA sequencing reagent (BigDye Terminator Cycle Sequencing FS Ready Reaction Kit). Thereafter, the DNA base sequence was analyzed with a DNA sequencer (ABI PRISM 3700, manufactured by Biosystems), and the obtained data was compiled into a database.
[0125]
The full length ratio of the 5′-end of the human cDNA library prepared by the improved oligo cap method was determined by the following method. For all clones in which the 5′-terminal sequence matches the human known mRNA in the public database, the 5′-terminal extends longer than the known mRNA sequence in the public database, or the 5′-terminal is short but the translation initiation codon Was judged as “full length”, and when it did not contain a translation initiation codon, it was judged as “non-full length”. Based on this, the total length of the 5'-end
[Number of full-length clones / (number of full-length clones + number of non-full-length clones)]
Was calculated. As a result, the total length ratio of the 5′-end was about 90% for both cDNA libraries. From this result, it was found that the full length ratio of the 5′-end sequence of the clone from the human cDNA library obtained by the oligocap method was very high.
Example 2
cDNA clone terminal sequence analysis and full-length nucleotide sequence analysis clone selection
For the plasmid DNA of the clones obtained from each cDNA library, the nucleotide sequence of the 5 ′ end of the cDNA was converted into a DNA sequencing reagent (Dye Terminator Cycle Sequencing Cycle Deion FS Ready Reactor Cycle Sequencing Cycle Sequencing Cycle Sequencing Cycle Sequencing Cycle Sequencing Cycle Sequencing Cycle Sequencing The sequencing reaction was performed with a DNA sequencer (ABI PRISM 3700, manufactured by Biosystems) using a cycle sequencing FS Ready Reaction Kit (manufactured by Biosystems) according to the manual. The obtained data was made into a database.
[0126]
For the 5 ′ terminal sequence of the analyzed cDNA clone, a homology search by BLAST was performed on the data with the complete cds of GenBank and UniGene, and those that were identical to the human mRNA sequence were excluded. Next, clustering was performed. When the homology was 90% or more and the consensus sequence was 50 base pairs or more, they were regarded as the same group, and a group was formed. A clone having a longer 5′-side in the group was selected, and the 3 ′ end sequence of the selected clone was analyzed and obtained by the same method as the 5 ′ end sequence as necessary. The obtained terminal sequence data was analyzed, and clones that made contigs with the 5 ′ end and 3 ′ end sequences were excluded. Further, as described above, the homology search by BLAST was used to remove those that were identical to the human mRNA sequence. A clone for full-length base sequence analysis was obtained from the clone thus selected.
Example 3
Full-length nucleotide sequence analysis
The base sequence of full length cDNA was determined for each clone selected for full length base sequence analysis. The base sequence was determined mainly by the primer walking method by the dideoxy terminator method using custom synthetic DNA primers. That is, using a custom synthetic DNA primer, a sequencing reaction was performed according to the manual using a DNA sequencing reagent manufactured by Biosystem, and then a DNA base sequence was analyzed using a sequencer manufactured by the same company. In some cases, a DNA sequencer manufactured by Licor was also used. Next, from the determined full-length base sequence, the translation region into the protein was estimated and the amino acid sequence was determined. BRACE2047573 was selected from the BRACE cDNA library, and TESTTI2053667 was selected from the TESTI cDNA library.
Example 4
Selection of clones with kinase-like sequences
First, the nucleic acid sequence of the helix clone was converted into a peptide sequence using a standard genetic code, and a database consisting only of peptide sequences consisting of 200 amino acids or more successively was created. Using the hidden Markov model PF00069 (PFAM database) representing the kinase catalytic domain as a query, a homology search for all helix clones was attempted using Wise 2.0 (estededb). Only clones having an expected value (Expect) of 0.01 or less were selected. As a result, a kinase catalytic activity domain was detected in 196 clones.
NCBI for the resulting clone
Using BLAST, a homology search for protein databases [GenPept (NCBI), refseq (NCBI), Aageneseq (Derwent)] was performed. As a result, two clones, “C-BRACE2047573” and “C-TESTI2053667”, were selected as clones having a kinase-like structure and novel in sequence.
The alignment of the clone with the top hit gene as a result of the homology search between known clones is shown in FIG. 2 and FIG.
[0127]
It was speculated that “C-BRACE2047573” encodes a novel human protein in its sequence and functions as a protein kinase.
[0128]
It was speculated that “C-TESTI2053667” encodes a novel human protein in its sequence and functions as a protein kinase.
[0129]
Moreover, these genes were confirmed to be human genes by mapping on the human genome sequence as follows, and further, the transcription initiation signal was identified in the structure upstream of the first exon.
Human genome sequences were published on April 14, 2003 (chr1.fa to chr22.fa, chrX.fa, chrY.fa and unplaced # contigs.fa; ftp://ftp.ncbi.nih.gov/ Genomes / H # sapiens / April # 14 # 2003 /) was used. The DNA sequences of “C-BRACE2037573” and “C-TESTI2053667” were each subjected to mapping by homology search using NCBI BLAST. The mapping results are shown in Table 1 and Table 2, respectively.
[0130]
[Table 1]

[0131]
In order to specify the position of C-BRACE2047573 on the chromosome, BLAST search was performed on the human genome sequence database (Apr. 14, 2003) using SEQ ID NO: 1 as a query. The obtained results were manually rearranged so as not to conflict with the normally expected exon-intron boundary (GT-AG rule). As a result, reasonable agreement was obtained in all regions of SEQ ID NO: 1.
[0132]
[Table 2]

[0133]
A BLAST search was performed on the human genome sequence database (Apr. 14, 2003) using SEQ ID NO: 1 as a query in order to specify the position of C-TESTI2053667 on the chromosome. The obtained results were manually rearranged so as not to conflict with the normally expected exon-intron boundary (AG-GT rule). As a result, reasonable agreement was obtained in all regions of SEQ ID NO: 3.
4 and 5 show the positions of the TATA signal and the initiator (INR) signal around the transcription start point.
From the results of Examples 1 to 4, it was shown that the gene of the present invention encodes a human full-length protein.
Example 5
(1) The expression frequency analysis of “C-BRACE2047573” was carried out by analyzing primer sets 5 ′ (tgctcctgct gctctttgct / SEQ ID NO: 9) and 3 ′ (cctccttaga cagaagcccc aca / SEQ ID NO: 10), Clonetech Multiple Tissue cDNA (MTC (TM)) Panels Human I and Human II total 16 tissue-derived cDNA (heart, brain, placenta, lung, liver, muscle, kidney, pancreas, (Spleen, thymus, prostate, testis, ovary, small intestine, large intestine and peripheral leukocytes) were amplified by PCR. Using a polymerase (Takara Taq Polymerase, R001A; Takara Bio Inc.) as a DNA synthesizing enzyme, 2.5 ng of template cDNA, 0.3 μM of each primer, 0.1 μm each of dNTP, It adjusted so that it might become. Each reaction solution was subjected to heat denaturation at 92 ° C. for 1 minute, followed by a temperature cycle of 92 ° C. for 10 seconds and 58 ° C. for 10 seconds. -25 times for each of the standardization genes C-BRACE2047573 and C-TESTI2053667, //-> 43 times <! --- and 35 times //-> repeated amplification. Amplified products were separated by 8% acrylamide gel electrophoresis and photographed after ethidium bromide staining.
The results are shown in FIG.
(2) The expression frequency analysis of “C-TESTI2053667” was performed using its specific primer set 5 ′ (catttgctga gccccctcttt gt / SEQ ID NO: 11) and 3 ′ (gctgaccccca ctttctttgt gt / SEQ ID NO: 12). Multiple Tissue cDNA (MTC (TM)) A total of 16 tissue-derived cDNAs of Panels Human I and Human II were used as a template in the same manner as described above. However, the temperature cycle was repeated 35 times.
The results are shown in FIG.
(3) GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase) was amplified from each template cDNA as described above as a control gene. For amplification, GAPDH Forward Primer and GAPDH Reverse Primer included in TaqMan GAPDH Control Reagents (402869; Applied Biosystems Japan Co., Ltd.) were used and amplified under the same conditions as described above. However, the temperature cycle was 25 times.
The results are shown in FIG.
[0134]
[Sequence Listing]

[0135]
[Possibility of industrial use]
According to the present invention, a novel human protein kinase protein and a gene encoding the protein are provided. Regulation of the phosphorylation state of a protein by a kinase plays a central role in normal differentiation / proliferation of cells and physiological functions at the cellular level. Since the novel kinase phosphatase of the present invention is considered to be deeply involved in intracellular physiological functions, the protein of the present invention is useful as a drug target molecule in drug development. In addition, the drug that acts on the protein of the present invention is expected to be an effective drug that can regulate intracellular physiological functions more precisely than drugs represented by conventional receptor agonists and antagonists.
In particular, C-BRACE20475573 was strongly expressed in the placenta, thus suggesting that it is a novel splice variant that plays an important role in the developmental period, particularly from the origin of this clone in the cerebellar developmental stage. As a result, it is expected to be used particularly effectively for diagnosis and treatment of abnormalities in developmental stage.
C-TESTI2053667 was found particularly strongly in immune system tissues such as leukocytes, spleen and thymus, and some expression was observed in developmental related tissues such as testis and placenta, pancreas and lung. Therefore, this gene is expected to be particularly useful for diagnosis and treatment of developmental abnormalities and diagnosis and treatment of immune diseases.
[Brief description of the drawings]
1 shows the vector pME18SFL3 produced in Example 1. FIG.
FIG. 2 shows an alignment between C-BRACE2037573 and its homologous top hit gene by CLUSTALW. The homologous top hit gene was obtained by performing a BLAST search on each database [GenPept (NCBI), refseq (NCBI) and Aagenesseq (Derwent)] using the peptide sequence of SEQ ID NO: 2 as a query. In the figure, gi | 108646650 | ref is the sequence of the PKCζ gene obtained from refseq.
FIG. 3 shows an alignment between C-TESTI2053667 and its homologous top hit gene by CLUSTALW. The homologous top hit gene was obtained by performing a BLAST search on each database [GenPept (NCBI), refseq (NCBI) and Aagenesseq (Derwent)] using the peptide sequence of SEQ ID NO: 2 as a query. In the figure, gi | 107727264 | gb is the sequence of a gene with unknown function obtained from GenPept.
FIG. 4 examined the transcription initiation signal upstream of the first exon in the results of Table 3. In conjunction with FIG. The region from 27001 to 27820 of NT_077984.1 in fa is shown. The numbers at the top of the sequence in the figure indicate the positions of bases counted from 27001. The transcription start point predicted from the presence of the initiator sequence consensus sequence (C / TC / TANT / AC / TC / TC) and the AT-rich region upstream of −45 to −25 is Consistent with the 5 ′ end of SEQ ID NO: 1.
FIG. 5 examined the transcription initiation signal upstream of the first exon in the results of Table 3. As shown in FIG. The region from 27001 to 27820 of NT_077984.1 in fa is shown. The numbers at the top of the sequence in the figure indicate the positions of bases counted from 27001. The transcription start point predicted from the presence of the initiator sequence consensus sequence (C / TC / TANT / AC / TC / TC) and the AT-rich region upstream of −45 to −25 is Consistent with the 5 ′ end of SEQ ID NO: 1.
6 examined the transcription initiation signal upstream of the first exon in the results of Table 3. FIG. Together with FIG. 7, in the genome chr13. An area from 27760001 to 27760680 of NC — 000013.5 in fa is shown. The numbers at the top of the sequences in the figure indicate the positions of bases counted from 27760001. The transcription start site predicted from the presence of the initiator sequence consensus (C / TC / TANT / AC / TC / TC) and the AT-rich region located upstream of -31 is SEQ ID NO: 1. Consistent with the 5 'end.
7 examined the transcription initiation signal upstream of the first exon in the results of Table 3. FIG. Together with FIG. 6, in the genome chr13. An area from 27760001 to 27760680 of NC — 000013.5 in fa is shown. The numbers at the top of the sequences in the figure indicate the positions of bases counted from 27760001. The transcription start site predicted from the presence of the initiator sequence consensus (C / TC / TANT / AC / TC / TC) and the AT-rich region located upstream of -31 is SEQ ID NO: 1. Consistent with the 5 'end.
FIG. 8 shows the result of expression frequency analysis of the gene of the present invention as an electrophoretic image of a PCR amplification product. C-BRACE2047573 was strongly expressed in the placenta. C-TESTI2053667 was found particularly strongly in immune system tissues such as leukocytes, spleen and thymus, and some expression was observed in developmental related tissues such as testis and placenta, pancreas and lung.

Claims (9)

DNA according to any one of (a) to (d) below.
(A) A DNA encoding a protein containing a novel region from No. 1 to No. 189 in the amino acid sequence shown in Sequence Listing 2, or a DNA encoding a novel protein of Sequence Listing 4.
(B) DNA containing a first exon region from No. 1 to No. 668 shown in Sequence Listing 1 or a novel DNA containing a coding region shown in Sequence Listing 3.
(C) one or more amino acids in the amino acid sequence of the novel region in Sequence Listing 2 or Sequence Listing 4 have an amino acid sequence substituted, deleted, inserted and / or added; DNA encoding a functionally equivalent protein.
(D) a protein that is hybridized with a DNA having a novel region in Sequence Listing 1 or Sequence Listing 3 under stringent conditions and functionally equivalent to a protein comprising the amino acid sequence described in Sequence Listing 2 or Sequence Listing 4 DNA encoding A DNA encoding a protein and a partial peptide comprising the amino acid sequence described in Sequence Listing 2 or Sequence Listing 4, according to Claim 1. A protein or peptide encoded by the DNA according to claim 1 or 2. A vector into which the DNA according to claim 1 or 2 is inserted A host cell carrying the DNA according to claim 1 or 2 or the vector according to claim 4. Culturing the host cell according to claim 5, and recovering a protein expressed from the host cell or a culture supernatant thereof.
A method for producing the protein or peptide thereof according to claim 3. An antibody that binds to the protein of claim 3. A polynucleotide comprising at least 15 nucleotides complementary to DNA consisting of the novel region described in Sequence Listing 1 or Sequence Listing 3 or a complementary strand thereof according to claim 1. A method for screening a compound that binds to the protein according to claim 3,
(A) contacting the test sample with the protein or its partial peptide, (b) detecting the binding activity between the protein or its partial peptide and the test sample,
(C) selecting a compound having an activity of binding to the protein or a partial peptide thereof,
Including methods.

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