JP4353697B2 - Cancer preventive / therapeutic agent - Google Patents

Description

【００７２】
また、本明細書中で繁用される置換基、保護基および試薬を下記の記号で表記する。

【００７３】
本願明細書の配列表の配列番号は、以下の配列を示す。
〔配列番号：１〕
ＴＬＲ１のアミノ酸配列を示す。
〔配列番号：２〕
配列番号：１で表されるアミノ酸配列を有するＴＬＲ１をコードするＤＮＡの塩基配列を示す。
〔配列番号：３〕
ＴＬＲ１をコードする全長遺伝子を含むＤＮＡの塩基配列を示す。
〔配列番号：４〕
実施例２で用いられたアンチセンスの塩基配列を示す。
〔配列番号：５〕
実施例２で用いられたプライマーの塩基配列を示す。
〔配列番号：６〕
実施例２で用いられたプライマーの塩基配列を示す。
〔配列番号：７〕
実施例２で用いられたプライマーの塩基配列を示す。
〔配列番号：８〕
実施例３で用いられたプライマーの塩基配列を示す。
〔配列番号：９〕
実施例３で用いられたプライマーの塩基配列を示す。
〔配列番号：１０〕
実施例３で用いられたプライマーの塩基配列を示す。
〔配列番号：１１〕
実施例３で用いられたプライマーの塩基配列を示す。
〔配列番号：１２〕
実施例３で用いられたプライマーの塩基配列を示す。
〔配列番号：１３〕
実施例３で用いられたプライマーの塩基配列を示す。
〔配列番号：１４〕
実施例３でベクターに挿入したＤＮＡ断片の塩基配列を示す。
【００７４】
以下において、実施例により本発明をより具体的にするが、この発明はこれらに限定されるものではない。
【実施例】
実施例１
乳がん組織で特異的に発現亢進している遺伝子群を明らかにするため、乳がん組織２例、その他２３種類の正常組織から抽出されたtotal RNA（表１）を材料とし、oligonucleotide microarray (Human Genome U95A, U95B, U95C, U95D, U95E; Affymetrix社) を用いて遺伝子発現解析を行った。
実験方法は、Affymetrix社の実験手引き書（Expression analysis technical manual）に従った。その結果、乳がん組織にのみ、ＴＬＲ１遺伝子の発現が検出された。解析した全ての正常組織では、検出限界以下であった（表２）。
【００７５】
【表１】

【表２】

【００７６】
実施例２
乳がん特異的に発現が観察されたＴＬＲ１遺伝子のアポトーシスに及ぼす影響を解析するために、ＴＬＲ１アンチセンスオリゴヌクレオチド導入実験を行った。まず、配列番号：３で表される塩基配列に対するアンチセンス（配列番号：４）を設計後、phosphorothioate化オリゴヌクレオチドを合成し、ＨＰＬＣ精製して導入実験に用いた（Amersham Pharmacia Biotech）。コントロールオリゴヌクレオチドとしては配列番号：４で示される塩基配列のリバース配列（配列番号：５）を同様にphosphorothioate化し、ＨＰＬＣ精製して用いた（Amersham Pharmacia Biotech）。
被験細胞として乳がん細胞株ＭＤＡ−ＭＢ−２３１（In Vitro、14(11)巻、911-915頁、1978年、ATCCより購入）を用い、オリゴヌクレオチド導入前日に１８ｘ１０³個の細胞を２４ウェルプレート（Falcon社製）に播種した。オリゴヌクレオチド導入にはOligofectAMINE（Invitrogen社製）を使用し、そのプロトコールに従った。導入後７時間でRNeasy mini kit（Qiagen社製）のプロトコールに従ってＲＮＡを抽出し、ThermoScriptRT-PCR system（GIBCO BRL社製）を用いてｃＤＮＡを調製した。ＲＣＲ反応は、２種のプライマー（配列番号：６および配列番号：７）、およびQuantumRNA 18S Internal Standards (Ambion社製)の１８Ｓ ｒＲＮＡ用プライマーおよびコンペティマーをマニュアルに従い同時に添加してMultiplexPCRを行った。ＰＣＲ反応後、反応液を電気泳動し、エチジウムブロミドによって染色されるバンドの強弱をBioImageIQソフトウェア（GenomicSolutions社製）によって１８Ｓ ｒＲＮＡのバンド強度で補正後、比較した。
一方、アポトーシスに及ぼす影響はアンチセンスオリゴヌクレオチド導入後３日目にCell death detection ELISA（Roche社製）を用いてコントロールオリゴ導入サンプルと比較した。
その結果、アンチセンス導入後７時間でＴＬＲ１の発現（ＲＮＡ）はコントロール（リバース）オリゴヌクレオチド導入時（１００％）に比べ５５％に減少した。導入後３日目でアポトーシスはコントロール（１００％）に比べ１８６％に増加していた。
これより、ＴＬＲ１はがん特異的に発現しているだけでなく、がん細胞の増殖やアポトーシスに関与していることが明らかとなった。
【００７７】
実施例３
（１）ＴＬＲ１、ＭＤ２発現ベクターの作製
ＴＬＲ１の発現系を作製するためにＴＬＲ１の全長遺伝子のクローニングを行った。
２種のプライマー（配列番号：８および配列番号：９）を使用し、Marathon ready cDNAライブラリー（Clontech社製）を鋳型として、Ｐｆｕポリメラーゼ（Straragene製）添付のプロトコールに従ってＰＣＲを行った。ＰＣＲは、10xPfu buffer 2μl、10mM each dNTP mixture(Clontech製) 0.4μl、20mMに調製した上記2種のプライマー 各0.8μl、鋳型cDNA溶液 0.5μl、Pfuポリメラーゼを0.1μlを含む20μlの反応液を、94℃、2分の前処理後、94℃・30秒、55℃・30秒、72℃・3分の反応を42サイクルおこなった後に、72℃・7分反応させることによりおこなった。
ＰＣＲ反応後、生成物をアガロースゲル電気泳動で分離し、目的のバンドを切り出してpCR4Blunt-TOPO vector（Invitrogen社製）にクローニングした。挿入断片の塩基配列を確認した結果、ヒトＴＬＲ１塩基配列と一致していたため、さらに２種のプライマー（配列番号：１０および配列番号：１１）を用いて上記で得られたベクターを鋳型としてＰＣＲを行った。ＰＣＲは、10xPfu buffer 2μl、10mM each dNTP mixture(Clontech製) 0.4μl、50mMに調製した上記２種のプライマー 各0.2μl、鋳型cDNA溶液 0.5μl、Ｐｆｕポリメラーゼを0.4μlを含む20μlの反応液を、94℃、2分の前処理後、94℃・30秒、55℃・30秒、72℃・3分の反応を25サイクルおこなった後に、72℃・7分反応させることによりおこなった。Ｐｆｕポリメラーゼ（Straragene製）を用いてＰＣＲ反応後、pCR4-TOPO vector（Invitrogen社製）添付のプロトコールに従いＰＣＲ産物の両末端にＡを付加し、生成物をアガロースゲル電気泳動で分離し、目的のバンドを切り出してpCR4-TOPO vector（Invitrogen社製）にクローニングした。配列に誤りの無いことを確認後、制限酵素BclII（ニッポンジーン製）で消化し、同様にBclIIで処理したp3XFLAG-CMV-14（SIGMA製）にLigation kit ver.2（Takara製）を用いて挿入した。配列に誤りの無いことを再度確認し、得られたＴＬＲ１発現ベクターを以下の実験に用いた。
一方、２種のプライマー（配列番号：１２および配列番号：１３）を使用し、ヒト脾臓由来ｃＤＮＡライブラリー（Clontech社製）を鋳型として、ＫＯＤポリメラーゼ（東洋紡製）を用いてＰＣＲを行った。ＰＣＲは、10xKOD buffer 2μl、2.5mM each dNTP mixture(Clontech製) 1.6μl、50mMに調製した上記2種のプライマー 各0.2μl、鋳型cDNA溶液 1μl、KODポリメラーゼを0.2μlを含む20μlの反応液を、94℃、2分の前処理後、94℃・30秒、55℃・30秒、72℃・3分の反応を42サイクルおこなった後に、72℃・7分反応させることによりおこなった。
ＰＣＲ反応後、生成物をアガロースゲル電気泳動で分離し、目的のバンドを切り出し、制限酵素KpnI、NotIで消化後、同様の制限酵素で消化したpcDNA3.1/Hyg(+)（Invitrogen製）にライゲーションし、ＭＤ２発現ベクターを得た。このベクターへの挿入断片の塩基配列を配列番号：１４に示す。挿入断片の塩基配列を確認した結果、ヒトＭＤ２の塩基配列と一致していた。
【００７８】
（２）ＴＬＲ１のレポーター活性に及ぼす影響
次に、ＴＬＲ１のがん細胞における活性を見るために、上記（１）で得られた発現ベクターをNCI-H1299細胞（ATCCより購入）に導入して種々のレポーター活性に及ぼす影響を見た。
以下に示す発現ベクターのトランスフェクション前日にNCI-H1299株を2×10⁴個ずつ24ウェルプレート（Falcon製）に播種し、Fugene6（Roche製）を用いて添付のプロトコールに従いトランスフェクションを行った。上記実施例３（１）で得られたＴＬＲ１発現ベクターおよび対照として用いたpcDNA3.1ベクターは0.19μｇ／ウェル使用した。レポーターとしては分泌型アルカリフォスファターゼ遺伝子の上流にＡＰ１サイトまたはＮＦ−κＢサイトを有するベクター（いずれもClontech製）、または蛍ルシフェラーゼ遺伝子の上流にＩＳＲＥサイトを有するベクター（Clontech製）を0.19μg／ウェル、レポーター活性を補正するために用いたpRL-TK（東洋インキ製）は0.13μg／ウェルを用いた。レポーターである分泌型アルカリフォスファターゼ活性または蛍ルシフェラーゼ活性は、トランスフェクション後２日目にそれぞれGreat EscAPe SEAP Fluorescence Detection Kitキット（Clontech製）、ピッカジーンデュアルシーパンジー ダブル ルシフェラーゼ アッセイシステム（東洋インキ製）のプロトコールに従って測定した。pRL-TKによって発現される海シイタケルシフェラーゼ活性はピッカジーンデュアルシーパンジー ダブル ルシフェラーゼ アッセイシステム（東洋インキ製）のプロトコールに従って測定し、レポーター活性値を補正した。
その結果、ＴＬＲ１の発現により、ＡＰ１レポーター活性は対照として用いたpcDNA3.1ベクターに比して5.6倍、ＮＦ−κＢレポーター活性は3.3倍、ＩＳＲＥレポーター活性は1.7倍それぞれ亢進していた。
これより、ＴＬＲ１を発現させることによって、肺がん由来NCI-H1299株では特にリガンドを加えなくともＡＰ１シグナル、ＮＦ−κＢシグナル、ＩＳＲＥシグナルを作動させることがわかった。
したがって、これらのプロモーター−レポーターアッセイを行うことにより、ＴＬＲ１シグナル調節作用を有する化合物をスクリーニングできる。
【００７９】
（３）パクリタキセルによるアポトーシス誘導に対する影響
次にＴＬＲ１シグナル作動ががんに及ぼす効果を見るために、パクリタキセルによるアポトーシス誘導に対する影響を観察した。
発現ベクタートランスフェクション前日に、HCT116（ATCCより購入）を5×10⁵個、10cmディッシュ（Falcon製）に播種し、Fugene6（Roche製）のプロトコールに従ってトランスフェクションを行った。上記実施例３（１）で得られたＴＬＲ１発現ベクターおよびＭＤ２発現ベクターを、またはコントロールとして用いたpcDNA3.1ベクター（Invitrogen製）を、いずれもディッシュあたり11μgになるように表３に示した組み合わせで使用した。トランスフェクション後４時間でトリプシン処理によって細胞を回収し、3×10³個ずつ96ウェルプレート（Falcon製）に播種し、ＴＬＲ１のリガンドであるN-palmitoyl-S-dilauryl-glyceryl CSK₄（Pam₃CSK₄）（ペプチド研究所製）を添加（1μg/ml）および／または抗がん剤であるパクリタキセル（Sigma製）を添加（0.125 μM）し、アポトーシスへの影響を見ることとした。
96ウェルプレートで培養２日後に、Cell Death Detection ELISA plus kit（Roche製）を用いて、添付のプロトコールに従いアポトーシスを検出した。図１に示すように、パクリタキセルが存在すると非存在時よりも吸光度が高く、パクリタキセルによりアポトーシスが誘導されていることが確認された（白バー対灰色バー）。それに対し、Pam₃CSK₄を添加することにより、吸光度が低下していることから、ＴＬＲ１リガンド添加によって、アポトーシスが低下していることがわかった（灰色バー対斜線バー）。さらにはPam₃CSK₄を添加しなくともＴＬＲ１とＭＤ２をトランスフェクションすることによってパクリタキセルで誘導されるアポトーシスが減少していることがわかった（灰色バー対黒バー）。
このことから、ＴＬＲ１シグナルが、抗がん剤によるアポトーシスを減弱させる効果があることが明らかとなった。
【００８０】
【発明の効果】
本発明で用いられるタンパク質は、がんの診断マーカーであり、したがって、該タンパク質の活性を調節（好ましくは阻害）する化合物またはその塩、該タンパク質の遺伝子の発現を調節（好ましくは阻害）する化合物またはその塩、本発明の抗体、本発明のアンチセンスヌクレオチドなどは、例えば大腸がん、乳がん、肺がん、前立腺がん、食道がん、胃がん、肝臓がん、胆道がん、脾臓がん、腎がん、膀胱がん、子宮がん、卵巣がん、精巣がん、甲状腺がん、膵臓がん、脳腫瘍または血液腫瘍などのがんの予防・治療剤、アポトーシス促進剤などとして安全な医薬として使用することができ、抗がん剤に耐性を示すがんの治療剤としても使用できる。また、抗がん剤（例、パクリタキセルなど）への感受性を高めるため、既存の抗がん剤の用量を減らし、副作用を軽減すこともできる。
【００８１】
【配列表】

【図面の簡単な説明】
【図１】 パクリタキセルによるアポトーシス誘導に対する影響を調べた結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a preventive / therapeutic agent and diagnostic agent for cancer.
[0002]
[Prior art]
In cancer chemotherapy, the development of new anti-cancer drugs has improved the life-prolonging effect, and the number of cases toward healing is increasing. However, most anti-cancer agents currently used are highly cytotoxic, which damages DNA or inhibits cell division, and thus damages normal cells to some extent. Strong side effects often appear in the actively divided bone marrow.
In order to comprehensively analyze gene expression, a microarray method in which cDNA or oligonucleotide is immobilized has been developed, and a technique for finding a disease-specific change in gene expression has spread and its usefulness has been confirmed. For example, Affymetrix's GeneChip system is being used extensively in the diagnosis of diseases such as cancer and the discovery of drug discovery target genes.
When an antisense oligonucleotide is introduced into a cell, it hybridizes with RNA having a complementary sequence, induces RNA degradation by RNase H, and inhibits protein translation, or inhibits direct protein synthesis by hybridization. Also bring. Since it is possible to specifically suppress the target gene function, it is frequently used as a gene function analysis means, and some antisense oligonucleotides have been developed for clinical application.
Recently, it has been clarified that the Toll receptor works as a receptor involved in innate immunity in insects where there is no acquired immune mechanism. Toll-like receptors (Toll-like receptors; TLRs), which are homologous to the Toll receptor, are also present in mammals and are also considered to be involved in innate immunity. Nearly 10 TLRs have been cloned to date. For example, TLR2 and TLR6 have been reported to recognize lipoproteins and glycolipids, TLR4 to recognize lipopolysaccharide, TLR5 to recognize Flagellin, and TLR9 to recognize CpG DNA (Nature Immunology 2 (8 ), 675-680, 2001). TLR1 (Proc Natl Acad Sci USA 95 (2), 588-593, 1998) is involved in microbial product recognition in cooperation with TLR2 (Non-patent Document 1: J Immunol. 165 (12)). Vol. 7125-7132 (2000), and inhibition of the action of TLR2 and TLR6 heteroreceptors (Non-patent Document 2: J Immunol. 166 (1), 15-19, 2001) It has been reported. More recently, TLR1 knockout mice have been analyzed, and TLR1 has been reconfirmed to be synergistic with TLR2 in ligand recognition. At the same time, PLR is newly used as a TLR1 ligand. _Three CSK _Four (Non-patent Document 3: J Immunol. 169 (1), 10-14, 2002) and OspA (Non-patent Document 4: Nat Med. 8 (8), 878-884, 2002) has been reported. In addition, research on signal transduction factors is also proceeding with analysis on TLR2 and TLR4. For example, from the analysis of receptor-interacting serine / threonine kinase-2 (RIPK2) knockout mice, this kinase acts on signal transduction of TLR2 and TLR4. (Non-patent Document 5: Nature. 416 (6877), 190-194, 2002), however, knowledge about the TLR1 signal is very poor.
On the other hand, paclitaxel, which is frequently used as an anticancer agent, exhibits a lipopolysaccharide-like action on mouse macrophages, but Kawasaki et al. Found that this action is due to signals of TLR4 and MD2 (Non-patent Document 6: J Biol Chem. 275 (4), 2251-2254, 2000). However, it is reported that both TLR4 and mouse MD2 are necessary, and that MD2 is species-specific and that paclitaxel has no LPS-like action in human MD2. Most of these TLR1 gene studies are related to cells of the immune system, and there is no report on the relationship between TLR1 gene and cancer (solid cancer).
[Non-Patent Document 1]
J Immunol. 165 (12), 7125-7132, 2000
[Non-Patent Document 2]
J Immunol. 166 (1), 15-19, 2001
[Non-Patent Document 3]
J Immunol. 169 (1), 10-14, 2002
[Non-Patent Document 4]
Nat Med. 8 (8), 878-884, 2002
[Non-Patent Document 5]
Nature.416 (6877), 190-194, 2002
[Non-Patent Document 6]
J Biol Chem. 275 (4), 2251-2254, 2000
[0003]
[Problems to be solved by the invention]
Drugs that target molecules specifically expressed in cancer cells and can inhibit cancer cell growth or induce apoptosis are highly desired. In addition, effective treatments for existing anticancer drug-resistant cancers and drugs that increase sensitivity to existing anticancer agents from the viewpoint of reducing side effects are also eagerly desired.
[0004]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have found a gene whose expression is remarkably increased in cancer tissue, and as a result of further investigation based on this finding, the present invention It came to complete.
[0005]
That is, the present invention
(1) Cancer comprising a compound or a salt thereof that inhibits the activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof Preventive and therapeutic agents,
(2) It contains a compound or its salt that inhibits the expression of the gene of the protein or its partial peptide or its salt that contains the same or substantially the same amino acid sequence as SEQ ID NO: 1. Cancer prevention / treatment agents,
(3) a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof, or the An antisense nucleotide containing a portion,
(4) A preventive / therapeutic agent for cancer comprising the antisense nucleotide according to (3) above,
(5) A preventive / therapeutic agent for cancer comprising an antibody against a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof, or a salt thereof,
(6) Cancer is colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovary The preventive / therapeutic agent according to (1) to (5) above, which is cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor,
(7) a diagnostic agent for cancer comprising an antibody against a protein containing the same or substantially the same amino acid sequence as that represented by SEQ ID NO: 1, or a partial peptide thereof, or a salt thereof;
(8) a diagnostic agent for cancer comprising a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof,
(9) Cancer is colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovary The diagnostic agent according to (7) or (8) above, which is cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor,
(10) A prophylactic / therapeutic agent for cancer comprising a compound having a Toll-like receptor activity inhibitory action or a salt thereof,
(11) A prophylactic / therapeutic agent for cancer comprising a compound having a Toll-like receptor expression inhibitory action or a salt thereof,
(12) Screening for a prophylactic / therapeutic agent for cancer, comprising using a protein containing the same or substantially the same amino acid sequence as SEQ ID NO: 1, or a partial peptide thereof, or a salt thereof. Method,
(13) A prophylactic / therapeutic agent for cancer, comprising a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof. Screening kit,
(14) Screening for a prophylactic / therapeutic agent for cancer, comprising using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. Method,
(15) A cancer preventive / therapeutic agent characterized by comprising a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof. Screening kit,
(16) A prophylactic / therapeutic agent for cancer obtainable by using the screening method according to (12) or (14) or the screening kit according to (13) or (15),
(17) Induction of apoptosis comprising a compound containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof, or a salt thereof, or a salt thereof Agent,
(18) A compound or a salt thereof that inhibits the expression of a gene of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Apoptosis inducer,
(19) A method for screening an apoptosis-inducing agent, comprising using a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1, a partial peptide thereof, or a salt thereof,
(20) A method for screening an apoptosis-inducing agent, comprising using a DNA encoding a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof,
(21) An antibody comprising a compound or a salt thereof that inhibits the activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof, or a salt thereof. Agent sensitivity enhancer,
(22) A compound or a salt thereof that inhibits the expression of a gene of a protein or a partial peptide thereof or a salt thereof containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1. Anticancer drug sensitivity enhancer,
(23) a compound or a salt thereof that inhibits the activity of a protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 or a partial peptide thereof or a salt thereof with respect to a mammal; Or a method for preventing or treating cancer, comprising administering an effective amount of a compound or salt thereof that inhibits the expression of the gene of the protein or a partial peptide thereof or a salt thereof,
(24) The activity of a protein containing the same or substantially the same amino acid sequence represented by SEQ ID NO: 1, or a partial peptide thereof, or a salt thereof for producing a prophylactic / therapeutic agent for cancer Provided is a use of a compound or a salt thereof, or a compound or a salt thereof that inhibits the expression of a gene of the protein or a partial peptide thereof or a salt thereof.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
A protein containing the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 1 (hereinafter also referred to as the protein of the present invention or the protein used in the present invention) is human or warm blood Animal (eg, guinea pig, rat, mouse, chicken, rabbit, pig, sheep, cow, monkey, etc.) cells (eg, hepatocytes, splenocytes, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermis cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, obesity) Cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells, hepatocytes Or stromal cells, or progenitors of these cells, stem cells or cancer cells) or any tissue in which these cells exist, such as the brain, brain regions (eg, olfactory bulb, amygdala, basal sphere, hippocampus) , Thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, digestive tract (eg, large intestine, Small intestine), blood vessels, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, skeletal muscle, etc. Also good.
[0007]
The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 is about 50% or more, preferably about 60% or more, more preferably about 70% or more, with the amino acid sequence represented by SEQ ID NO: 1. More preferred is an amino acid sequence having a homology of about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more.
Examples of the protein containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 1. A protein having substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 1 is preferable.
[0008]
The substantially equivalent activities include, for example, signal transduction activity (eg, intracellular signal transduction activity of Toll-like receptor (preferably TLR1)), ligand binding activity (eg Toll-like receptor (preferably TLR1)) And the like, and the like. Substantially homogeneous indicates that their properties are qualitatively (eg, physiologically or pharmacologically) homogeneous. Therefore, it is preferable that the signal transduction activity and the ligand binding activity are equal (eg, about 0.01 to 100 times, preferably about 0.1 to 10 times, more preferably 0.5 to 2 times). However, quantitative factors such as the degree of these activities and the molecular weight of the protein may be different.
Examples of the ligand include bacterial-derived lipopeptides (preferably triacylated lipopeptides), or N-palmitoyl-S-dipalmitoylglyceryl (Pam _Three ) Cys- Ser- (Lys) _Four (CSK _Four ), N-palmitolyl-S-dilaurylgryceryl (N-Pam-S-Lau ₂ ) CSK _Four Triacyl peptides such as Borrelia burgdorferi and outer-surface lipoprotein (OspA) are used. Signal transduction activity and ligand binding activity can be measured according to a known method, for example, the method described in J. Immunol. 166 (1), pages 15-19, 2001 or a method analogous thereto. The TLR family activates the NF-κB signal by ligand binding. Thus, for example, the activity of a protein [eg, Toll-like receptor (eg, TLR1)] used in the present invention is expressed by a reporter gene containing an expression vector of the protein in an NF-κB binding sequence in a promoter or enhancer. (A) microbial cell lysate, microbial culture supernatant, eukaryotic cell lysate, eukaryotic cell culture supernatant and other liquids containing ligand, (b) ligand itself or ( c) A substance having a binding activity equivalent to that of a natural ligand is added, and the expression level of the reporter is measured by a known method. The amount of reporter expression can be measured, for example, using reporter protein activity using luciferase activity, alkaline phosphatase activity, or the like as an index.
In addition, the signal transduction activity can be measured by measuring and comparing the apoptotic activity against anticancer agents (eg, paclitaxel, etc.) in the presence or absence of expression of proteins (eg, TLR1 etc.) used in the present invention. . Apoptotic activity against an anticancer drug (eg, paclitaxel, etc.) in the presence and absence of a ligand can be measured and compared by a known method to measure signal transduction activity.
Furthermore, in order to measure the signal transduction of TLR1 used in the present invention with higher sensitivity, a cell having the ability to express a TLR1 signal-related molecule (eg, TLR2, MD2, etc.) may be used. The above activity may be measured by forcibly expressing a TLR1 signal-related molecule (eg, TLR2, MD2, etc.).
[0009]
Examples of the protein used in the present invention include (1) 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 30 in the amino acid sequence represented by SEQ ID NO: 1, preferably Is an amino acid sequence from which about 1 to 10, more preferably a number (1 to 5) amino acids have been deleted. (2) 1 or 2 or more (for example, 1 to 2) in the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence added with about 100, preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5) amino acids, (3) represented by SEQ ID NO: 1. An amino acid in which one or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably several (1 to 5)) amino acids are inserted into the amino acid sequence Array, ▲ 4 1 or 2 or more (for example, about 1 to 100, preferably about 1 to 30, preferably about 1 to 10, more preferably a number (1 to 5) in the amino acid sequence represented by SEQ ID NO: 1. And so-called muteins such as proteins containing amino acid sequences in which amino acids are substituted with other amino acids, or (5) amino acid sequences obtained by combining them.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited.
[0010]
The protein in the present specification has an N-terminus (amino terminus) at the left end and a C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. The protein used in the present invention, including the protein containing the amino acid sequence represented by SEQ ID NO: 1, has a carboxyl group (—COOH), carboxylate (—COO) at the C-terminus. ^- ), Amide (-CONH ₂ ) Or ester (—COOR).
Here, as R in the ester, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. _1-6 Alkyl groups such as C, such as cyclopentyl, cyclohexyl, etc. _3-8 A cycloalkyl group such as phenyl, α-naphthyl and the like _6-12 Aryl groups such as phenyl-C such as benzyl, phenethyl and the like _1-2 Alkyl groups or α-naphthyl-C such as α-naphthylmethyl _1-2 C such as alkyl group _7-14 Aralkyl groups, pivaloyloxymethyl groups and the like are used.
When the protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. As the ester in this case, for example, the above C-terminal ester or the like is used.
Further, in the protein used in the present invention, the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected by a protecting group (for example, a C-form such as formyl group, acetyl group, etc.). _1-6 C such as alkanoyl _1-6 A group protected by an acyl group, an N-terminal glutamine residue produced by cleavage in vivo, pyroglutamine oxidized, a substituent on the side chain of an amino acid in the molecule (eg, —OH, —SH) , Amino group, imidazole group, indole group, guanidino group, etc.) are suitable protecting groups (for example, C such as formyl group, acetyl group, etc.) _1-6 C such as alkanoyl group _1-6 Examples thereof include those protected with an acyl group or the like, or complex proteins such as so-called glycoproteins to which sugar chains are bound.
Specific examples of the protein used in the present invention include a protein containing the amino acid sequence represented by SEQ ID NO: 1 (TLR1).
[0011]
The partial peptide of the protein used in the present invention is a partial peptide of the protein used in the present invention as described above, and preferably any one having the same properties as the protein used in the present invention described above. It may be a thing.
Specifically, for the purpose of preparing the antibody of the present invention described later, a peptide having the 27th to 578th amino acid sequence in the amino acid sequence represented by SEQ ID NO: 1 and the like can be mentioned. For example, it has at least 20 or more, preferably 50 or more, more preferably 70 or more, more preferably 100 or more, most preferably 200 or more amino acid sequences of the constituent amino acid sequences of the protein used in the present invention. Peptides are used.
In addition, the partial peptide used in the present invention has one or more (preferably about 1 to 10, more preferably several (1 to 5)) amino acids in its amino acid sequence deleted, or 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are added to the amino acid sequence, or the amino acid 1 or 2 or more (preferably about 1 to 20, more preferably about 1 to 10, more preferably several (1 to 5)) amino acids are inserted into the sequence, or in the amino acid sequence One or more amino acids (preferably about 1 to 10, more preferably about several, and more preferably about 1 to 5) may be substituted with other amino acids.
[0012]
In the partial peptide used in the present invention, the C-terminus is carboxyl group (—COOH), carboxylate (—COO). ^- ), Amide (-CONH ₂ ) Or ester (—COOR).
Furthermore, the partial peptide used in the present invention includes those having a carboxyl group (or carboxylate) in addition to the C terminus, as well as the N-terminal amino acid residue (eg, the protein used in the present invention described above). , The amino group of the methionine residue) is protected with a protecting group, the N-terminal side is cleaved in vivo and the glutamine residue produced is pyroglutamine oxidized, the substituent on the side chain of the amino acid in the molecule is Also included are those protected with an appropriate protecting group, or complex peptides such as so-called glycopeptides linked to sugar chains.
The partial peptide used in the present invention can also be used as an antigen for antibody production.
[0013]
As a salt of a protein or partial peptide used in the present invention, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or base (eg, alkali metal salt) is used. The acid addition salts that are acceptable are preferred. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.
The protein used in the present invention or a partial peptide thereof or a salt thereof can be produced from the aforementioned human or warm-blooded animal cells or tissues by a known protein purification method, or can include a trait containing DNA encoding the protein. It can also be produced by culturing the transformant. Moreover, it can also manufacture according to the below-mentioned peptide synthesis method.
When producing from human or mammalian tissue or cells, the human or mammalian tissue or cells are homogenized and then extracted with acid, etc., and the extract is subjected to chromatography such as reverse phase chromatography or ion exchange chromatography. Can be purified and isolated.
[0014]
For the synthesis of the protein or partial peptide used in the present invention or a salt thereof, or an amide thereof, a commercially available resin for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. it can. Using such a resin, an amino acid in which the α-amino group and the side chain functional group are appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target protein. At the end of the reaction, the protein or partial peptide is excised from the resin, and at the same time, various protecting groups are removed, and further, intramolecular disulfide bond forming reaction is performed in a highly diluted solution to obtain the target protein or partial peptide or their amides. To do.
For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for protein synthesis can be used, and carbodiimides are particularly preferable. As the carbodiimide, DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like are used. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (for example, HOBt, HOBt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. It can later be added to the resin.
[0015]
The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for protein condensation reactions. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane, and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range that is known to be usable for protein bond forming reactions, and is usually selected from the range of about -20 ° C to 50 ° C. The activated amino acid derivative is usually used in an excess of 1.5 to 4 times. As a result of a test using the ninhydrin reaction, if the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even if the reaction is repeated, acetylation of the unreacted amino acid with acetic anhydride or acetylimidazole can prevent the subsequent reaction from being affected.
[0016]
Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.
The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (for example, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.
The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of the group suitable for esterification include, for example, lower (C _1-6 ) Aroyl groups such as alkanoyl group and benzoyl group, groups derived from carbonic acid such as benzyloxycarbonyl group and ethoxycarbonyl group are used. Examples of the group suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a t-butyl group.
Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, Br-Z, t-butyl and the like are used.
Examples of the protecting group for imidazole of histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, Fmoc, and the like.
[0017]
Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.
Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4 Addition of a cation scavenger such as -butanedithiol, 1,2-ethanedithiol, etc. is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.
[0018]
The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the removal of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.
As another method for obtaining an amide form of a protein or partial peptide, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, and then the peptide (protein) chain is formed on the amino group side to the desired chain length. After the extension, a protein or partial peptide from which only the N-terminal α-amino group protecting group of the peptide chain is removed and a protein or partial peptide from which only the C-terminal carboxyl group protecting group has been removed are produced, and these The protein or peptide is condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected protein or peptide obtained by condensation, all the protecting groups can be removed by the above method to obtain the desired crude protein or peptide. This crude protein or peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide form of the desired protein or peptide.
In order to obtain a protein or peptide ester, for example, the α-carboxyl group of the carboxy-terminal amino acid is condensed with a desired alcohol to form an amino acid ester, and then the desired protein or peptide amide is obtained in the same manner as the protein or peptide amide. An ester of a peptide can be obtained.
[0019]
The partial peptide used in the present invention or a salt thereof can be produced according to a known peptide synthesis method or by cleaving the protein used in the present invention with an appropriate peptidase. As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, a partial peptide or amino acid that can constitute the partial peptide used in the present invention is condensed with the remaining portion, and when the product has a protective group, the target peptide can be produced by removing the protective group. it can. Examples of known condensation methods and protecting group removal include the methods described in (1) to (5) below.
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(5) Supervised by Yajima Haruaki, Development of follow-up medicine, Volume 14, Peptide synthesis, Hirokawa Shoten
Further, after the reaction, the partial peptide used in the present invention can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like. When the partial peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method similar thereto, and conversely, when obtained as a salt, the known method or a method equivalent thereto. It can be converted to the free form or other salts by methods.
[0020]
The polynucleotide encoding the protein used in the present invention may be any as long as it contains the base sequence encoding the protein used in the present invention described above. Preferably it is DNA. The DNA may be any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA.
The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like. Moreover, it can also be directly amplified by reverse transcriptase polymerase chain reaction (hereinafter abbreviated as RT-PCR method) using a total RNA or mRNA fraction prepared from the cells / tissues described above.
Examples of the DNA encoding the protein used in the present invention include a DNA containing the base sequence represented by SEQ ID NO: 2 or a hybrid with the base sequence represented by SEQ ID NO: 2 under highly stringent conditions. Any DNA may be used as long as it encodes a protein containing a soybean base sequence and having substantially the same quality as the protein containing the amino acid sequence represented by SEQ ID NO: 1.
[0021]
Examples of DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 under highly stringent conditions include, for example, about 50% or more, preferably about 60% or more, with the base sequence represented by SEQ ID NO: 2. More preferably, DNA containing a base sequence having a homology of about 70% or more, more preferably about 80% or more, particularly preferably about 90% or more, and most preferably about 95% or more is used.
Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual. More preferably, it can be carried out according to highly stringent conditions.
The highly stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. In particular, the case where the sodium concentration is about 19 mM and the temperature is about 65 ° C. is most preferable.
More specifically, as the DNA encoding the protein containing the amino acid sequence represented by SEQ ID NO: 1, DNA containing the base sequence represented by SEQ ID NO: 2 or the like is used.
[0022]
The DNA encoding the partial peptide used in the present invention may be any DNA as long as it contains a base sequence encoding the partial peptide used in the present invention described above. Further, any of genomic DNA, genomic DNA library, cDNA derived from the cells / tissues described above, cDNA library derived from the cells / tissues described above, and synthetic DNA may be used.
Examples of the DNA encoding the partial peptide used in the present invention include DNA having a part of DNA containing the base sequence represented by SEQ ID NO: 2, or the base sequence represented by SEQ ID NO: 2 and high string A DNA containing a part of a DNA encoding a protein that contains a base sequence that hybridizes under a gentle condition and has substantially the same activity as the protein of the present invention is used.
The DNA that can hybridize with the base sequence represented by SEQ ID NO: 2 has the same significance as described above.
Hybridization methods and highly stringent conditions are the same as those described above.
[0023]
Means for cloning DNA that completely encodes the protein and partial peptide used in the present invention (hereinafter, in the description of cloning and expression of DNA encoding them, these may be simply abbreviated as the protein of the present invention) As described above, a synthetic DNA primer having a part of the base sequence encoding the protein of the present invention is used to amplify by PCR method, or DNA incorporated into an appropriate vector encodes a part or all region of the protein of the present invention. Can be selected by hybridization with a DNA fragment or synthetic DNA labeled. The hybridization method can be performed according to the method described in Molecular Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989), for example. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.
DNA base sequence conversion is performed by PCR, a known kit such as Mutan. ^TM -super Express Km (Takara Shuzo Co., Ltd.), Mutan ^TM -K (Takara Shuzo Co., Ltd.) can be used according to a known method such as ODA-LA PCR method, Gapped duplex method, Kunkel method or the like, or a method analogous thereto.
The DNA encoding the cloned protein can be used as it is or after digestion with a restriction enzyme or addition of a linker if desired. The DNA may have ATG as a translation initiation codon on the 5 ′ end side, and may have TAA, TGA or TAG as a translation termination codon on the 3 ′ end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
The protein expression vector of the present invention is, for example, (a) excising the target DNA fragment from the DNA encoding the protein of the present invention, and (b) ligating the DNA fragment downstream of the promoter in an appropriate expression vector. Can be manufactured.
[0024]
Examples of vectors include E. coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, and the like. In addition to animal viruses such as bacteriophage, retrovirus, vaccinia virus and baculovirus, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like are used.
The promoter used in the present invention may be any promoter as long as it is suitable for the host used for gene expression. For example, when animal cells are used as a host, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and the like can be mentioned.
Of these, the CMV (cytomegalovirus) promoter, SRα promoter and the like are preferably used. When the host is Escherichia, trp promoter, lac promoter, recA promoter, λP _L When the host is a Bacillus genus, the promoter, lpp promoter, T7 promoter, etc., SPO1 promoter, SPO2 promoter, penP promoter, etc. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. Is preferred. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.
[0025]
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin (hereinafter sometimes abbreviated as SV40ori) and the like is used as desired. it can. Examples of selection markers include dihydrofolate reductase (hereinafter sometimes abbreviated as dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance gene (hereinafter Amp). ^r A neomycin resistance gene (hereinafter Neo) ^r G418 resistance) and the like. In particular, when a dhfr gene-deficient Chinese hamster cell is used and the dhfr gene is used as a selection marker, the target gene can also be selected by a medium not containing thymidine.
If necessary, a signal sequence suitable for the host is added to the N-terminal side of the protein of the present invention. When the host is Escherichia, PhoA signal sequence, OmpA, signal sequence, etc., and when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc. are used in yeast. In some cases, MFα • signal sequence, SUC2 • signal sequence, etc. When the host is an animal cell, insulin signal sequence, α-interferon signal sequence, antibody molecule / signal sequence, etc. can be used.
A transformant can be produced using a vector containing the DNA encoding the protein of the present invention thus constructed.
[0026]
As the host, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, insects, animal cells and the like are used.
Specific examples of the genus Escherichia include, for example, Escherichia coli K12 • DH1 [Procedures of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981)], JA221 [Journal of Molecular Biology (Journal of Molecular Biology). Biology), 120, 517 (1978)], HB101 [Journal of Molecular Biology, 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc. Used.
Examples of Bacillus bacteria include, for example, Bacillus subtilis MI114 [Gen, 24, 255 (1983)], 207-21 [Journal of Biochemistry, 95, 87 (1984). )] Etc. are used.
Examples of yeast include Saccharomyces cerevisiae AH22, AH22R. ^- NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris KM71, and the like.
[0027]
As insect cells, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five derived from eggs of Trichoplusia ni ^TM Cells, cells derived from Mamestra brassicae or cells derived from Estigmena acrea are used. When the virus is BmNPV, sputum-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217, (1977)).
As insects, for example, silkworm larvae are used [Maeda et al., Nature, 315, 592 (1985)].
Examples of animal cells include monkey cell COS-7, Vero, Chinese hamster cell CHO (hereinafter abbreviated as CHO cell), dhfr gene-deficient Chinese hamster cell CHO (hereinafter referred to as CHO (dhfr). ^- Abbreviated cells), mouse L cells, mouse AtT-20, mouse myeloma cells, mouse ATDC5 cells, rat GH3, human FL cells and the like.
To transform Escherichia, for example, Proc. Natl. Acad. Sci. USA, 69, 2110 (Proc. Natl. Acad. Sci. USA) 1972), Gene, Vol. 17, 107 (1982), and the like.
[0028]
Transformation of Bacillus can be performed, for example, according to the method described in Molecular & General Genetics, 168, 111 (1979).
To transform yeast, for example, Methods in Enzymology, 194, 182-187 (1991), Processings of the National Academy of Sciences of -The method can be carried out according to the method described in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
Insect cells or insects can be transformed, for example, according to the method described in Bio / Technology, 6, 47-55 (1988).
In order to transform animal cells, for example, Cell Engineering Supplement 8 New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973).
In this way, a transformant transformed with an expression vector containing DNA encoding the protein can be obtained.
When cultivating a transformant whose host is an Escherichia bacterium or Bacillus genus, a liquid medium is suitable as a medium used for the culture, including a carbon source necessary for the growth of the transformant, Nitrogen sources, inorganic substances, etc. are contained. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose. Examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean cake, and potato extract. Examples of inorganic or organic substances and inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. In addition, yeast extract, vitamins, growth promoting factors and the like may be added. The pH of the medium is preferably about 5-8.
[0029]
As a medium for culturing Escherichia, for example, M9 medium containing glucose and casamino acid [Miller, Journal of Experiments in Molecular Genetics] 431-433, Cold Spring Harbor Laboratory, New York 1972]. In order to make the promoter work efficiently as necessary, a drug such as 3β-indolylacrylic acid can be added.
When the host is an Escherichia bacterium, the culture is usually carried out at about 15 to 43 ° C. for about 3 to 24 hours, and if necessary, aeration or agitation can be added.
When the host is a genus Bacillus, the culture is usually performed at about 30 to 40 ° C. for about 6 to 24 hours, and if necessary, aeration or agitation can be added.
When cultivating a transformant whose host is yeast, examples of the medium include a Burkholder minimum medium [Bostian, KL et al., Proceedings of the National Academy of Sciences of Science. The USA (Proc. Natl. Acad. Sci. USA), 77, 4505 (1980)] and SD medium containing 0.5% casamino acid [Bitter, GA et al., Proceedings of the National -Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA), 81, 5330 (1984)]. The pH of the medium is preferably adjusted to about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours, and aeration and agitation are added as necessary.
When culturing a transformant whose host is an insect cell or an insect, a medium such as 10% bovine serum immobilized in Grace's Insect Medium (Grace, TCC, Nature, 195,788 (1962)) is added. What added the thing suitably is used. The pH of the medium is preferably adjusted to about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days, and aeration and agitation are added as necessary.
When cultivating a transformant whose host is an animal cell, examples of the medium include a MEM medium containing about 5 to 20% fetal bovine serum [Science, Vol. 122, 501 (1952)], DMEM medium. [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)] is used. The pH is preferably about 6-8. Culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours, and aeration and agitation are added as necessary.
As described above, the protein of the present invention can be produced inside the cell, the cell membrane, or outside the cell of the transformant.
[0030]
Separation and purification of the protein of the present invention from the culture can be performed, for example, by the following method.
When extracting the protein of the present invention from cultured cells or cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freeze-thaw, etc. For example, a method of obtaining a crude protein extract by centrifugation or filtration after destroying cells or cells by the method is appropriately used. Protein denaturants such as urea and guanidine hydrochloride in the buffer, Triton X-100 ^TM A surfactant such as may be contained. When the protein is secreted into the culture solution, after completion of the culture, the cells or cells and the supernatant are separated by a known method, and the supernatant is collected.
Purification of the protein contained in the thus obtained culture supernatant or extract can be performed by appropriately combining known separation / purification methods. These known separation and purification methods include mainly molecular weights such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Method using difference in charge, method using difference in charge such as ion exchange chromatography, method using specific affinity such as affinity chromatography, and difference in hydrophobicity such as reverse phase high performance liquid chromatography A method using a difference in isoelectric point, such as a method, isoelectric focusing method, or the like is used.
[0031]
When the protein thus obtained is obtained in a free form, it can be converted into a salt by a known method or a method analogous thereto, and conversely, when obtained as a salt, by a known method or a method analogous thereto, It can be converted to the free form or other salts.
The protein produced by the recombinant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act on the protein before or after purification. Examples of protein modifying enzymes include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of the protein of the present invention thus produced can be measured by enzyme immunoassay or Western blotting using a specific antibody.
[0032]
The antibody to the protein or partial peptide or salt thereof used in the present invention may be either a polyclonal antibody or a monoclonal antibody as long as it can recognize the protein or partial peptide or salt thereof used in the present invention.
An antibody against the protein or partial peptide used in the present invention or a salt thereof (hereinafter, in the description of the antibody, these may be simply abbreviated as the protein of the present invention) is known using the protein of the present invention as an antigen. The antibody or antiserum can be produced according to the production method.
[Production of monoclonal antibodies]
(A) Preparation of monoclonal antibody-producing cells
The protein of the present invention is administered to a warm-blooded animal by itself, together with a carrier or a diluent, at a site where antibody production is possible by administration. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 2 to 6 weeks, about 2 to 10 times in total. Examples of the warm-blooded animal used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens, and mice and rats are preferably used.
When producing monoclonal antibody-producing cells, select a warm-blooded animal immunized with an antigen, for example, an individual with a confirmed antibody titer from a mouse, and collect spleen or lymph nodes 2 to 5 days after the final immunization. Monoclonal antibody-producing hybridomas can be prepared by fusing the antibody-producing cells to be fused with allogeneic or xenogeneic myeloma cells. The antibody titer in the antiserum can be measured, for example, by reacting the labeled protein described below with the antiserum and then measuring the activity of the labeling agent bound to the antibody. The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used.
[0033]
Examples of myeloma cells include warm-blooded animal myeloma cells such as NS-1, P3U1, SP2 / 0, AP-1, and P3U1 is preferably used. The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of myeloma cells used is about 1: 1 to 20: 1, and PEG (preferably PEG 1000 to PEG 6000) is added at a concentration of about 10 to 80%. Cell fusion can be efficiently carried out by incubating at 20 to 40 ° C., preferably 30 to 37 ° C. for 1 to 10 minutes.
Various methods can be used to screen for monoclonal antibody-producing hybridomas. For example, the hybridoma culture supernatant is added to a solid phase (eg, microplate) on which a protein antigen is adsorbed directly or together with a carrier, and then a radioactive substance or An anti-immunoglobulin antibody labeled with an enzyme or the like (when the cell used for cell fusion is a mouse, an anti-mouse immunoglobulin antibody is used) or protein A, and a method for detecting a monoclonal antibody bound to a solid phase; Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which a globulin antibody or protein A is adsorbed, a protein labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected.
The selection of the monoclonal antibody can be performed according to a known method or a similar method. Usually, it can be performed in a medium for animal cells supplemented with HAT (hypoxanthine, aminopterin, thymidine). As the selection and breeding medium, any medium may be used as long as it can grow a hybridoma. For example, RPMI 1640 medium containing 1-20%, preferably 10-20% fetal calf serum, GIT medium (Wako Pure Chemical Industries, Ltd.) containing 1-10% fetal calf serum, or serum-free for hybridoma culture A culture medium (SFM-101, Nissui Pharmaceutical Co., Ltd.) etc. can be used. The culture temperature is usually 20 to 40 ° C, preferably about 37 ° C. The culture time is usually 5 days to 3 weeks, preferably 1 week to 2 weeks. Culturing can usually be performed under 5% carbon dioxide gas. The antibody titer of the hybridoma culture supernatant can be measured in the same manner as the antibody titer in the above antiserum.
[0034]
(B) Purification of monoclonal antibody
Monoclonal antibodies can be separated and purified by known methods such as immunoglobulin separation and purification methods (eg, salting-out method, alcohol precipitation method, isoelectric point precipitation method, electrophoresis method, ion exchanger (eg, DEAE)). Desorption method, ultracentrifugation method, gel filtration method, antigen-binding solid phase or a specific purification method for obtaining an antibody by dissociating the binding using an active adsorbent such as protein A or protein G. it can.
[Preparation of polyclonal antibody]
The polyclonal antibody of the present invention can be produced according to a known method or a method analogous thereto. For example, an immune antigen (protein antigen) itself or a complex of it and a carrier protein is prepared, and a warm-blooded animal is immunized in the same manner as the above-described monoclonal antibody production method, and an antibody against the protein of the present invention is contained from the immunized animal. The product can be produced by collecting and purifying the antibody.
Regarding the complex of immunizing antigen and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio of carrier and hapten are such that antibodies are more effective against hapten immunized by cross-linking to carrier. As long as it is possible, any substance may be cross-linked at any ratio. For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to 20, preferably about 1 with respect to hapten 1 by weight ratio. A method of coupling at a rate of ˜5 is used.
Various coupling agents can be used for coupling of the hapten and the carrier, but active ester reagents containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, and dithiobilidyl group are used.
The condensation product is administered to a warm-blooded animal by itself, together with a carrier and a diluent, at a site where antibody production is possible. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total.
Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of warm-blooded animals immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.
[0035]
Complementary or substantially complementary to the base sequence of DNA encoding the protein or partial peptide used in the present invention (hereinafter, in the description of antisense nucleotide, these DNAs may be abbreviated as the DNA of the present invention). An antisense nucleotide having a nucleotide sequence complementary to or a part thereof includes a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the DNA of the present invention, Any antisense nucleotide may be used as long as it has an action capable of suppressing expression, but antisense DNA is preferred.
The base sequence substantially complementary to the DNA of the present invention is, for example, about 70 of the total base sequence or partial base sequence of the base sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). %, Preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more of a base sequence having a homology. In particular, among the entire base sequences of the complementary strand of the DNA of the present invention, (a) in the case of an antisense nucleotide directed to translation inhibition, the base sequence of the portion encoding the N-terminal site of the protein of the present invention (for example, the start codon) An antisense nucleotide having a homology of about 70% or more, preferably about 80% or more, more preferably about 90% or more, and most preferably about 95% or more with a complementary strand of a nearby base sequence) (b) In the case of an antisense nucleotide directed to RNA degradation by RNase H, it is about 70% or more, preferably about 80% or more, more preferably about 90% or more, most complementary to the complementary strand of the entire base sequence of the DNA of the present invention including introns. Antisense nucleotides having a homology of about 95% or more are preferred respectively.
Specifically, an antisense polynucleotide having a base sequence complementary to or substantially complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3, or a part thereof Preferably, for example, an antisense polynucleotide having a base sequence complementary to the base sequence of DNA containing the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 or a part thereof (more preferably, SEQ ID NO: 2 Or an antisense polynucleotide having a base sequence complementary to the base sequence of the DNA containing the base sequence represented by SEQ ID NO: 3 or a part thereof.
The antisense polynucleotide is usually composed of about 10 to 40 bases, preferably about 15 to 30 bases.
In order to prevent degradation by a hydrolase such as nuclease, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are changed to chemically modified phosphate residues such as phosphorothioate, methylphosphonate and phosphorodithionate. May be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base portion (pyrimidine, purine) is also chemically modified. Any one may be used as long as it hybridizes with DNA having the base sequence represented by SEQ ID NO: 2. These antisense nucleotides can be produced using a known DNA synthesizer.
[0036]
According to the present invention, an antisense nucleotide (nucleic acid) capable of inhibiting the replication or expression of the protein gene of the present invention is designed based on the base sequence information of the DNA encoding the cloned or determined protein. Can be synthesized. Such a polynucleotide (nucleic acid) can hybridize with the RNA of the protein gene of the present invention, inhibit the synthesis or function of the RNA, or through interaction with the protein-related RNA of the present invention. Thus, the expression of the protein gene of the present invention can be regulated and controlled. The polynucleotide complementary to the selected sequence of the protein-related RNA of the present invention and the polynucleotide capable of specifically hybridizing with the protein-related RNA of the present invention are the protein gene of the present invention in vivo and in vitro. It is useful for regulating / controlling the expression of and for treating or diagnosing diseases and the like. The term “corresponding” means homologous to or complementary to a specific sequence of nucleotides, base sequences or nucleic acids including genes. “Corresponding” between a nucleotide, base sequence or nucleic acid and peptide (protein) usually refers to the amino acid of the peptide (protein) in the instruction derived from the nucleotide (nucleic acid) sequence or its complement. . 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' The end palindromic region and the 3 ′ end hairpin loop can be selected as the preferred region of interest, but any region within the protein gene can be selected as the subject.
Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize to the target region, the target nucleic acid It can be said that it is “antisense”. Antisense (poly) nucleotides are polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases Or other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymers are found in DNA or RNA And a nucleotide having a configuration allowing base attachment). They can be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, and also DNA: RNA hybrids, and unmodified polynucleotides (or unmodified oligonucleotides), and more publicly known Modified, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotides Modified, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged or sulfur-containing bonds (eg, phosphorothioates, phosphorodithioates, etc.) ), For example, proteins (nucleases, nuclease inhibitors, and toxins) , Antibodies, signal peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelates Compounds containing compounds (for example, metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those having modified bonds (eg, α-anomeric nucleic acids, etc.) It may be. Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.
The antisense (poly) nucleotide (nucleic acid) of the present invention is RNA, DNA, or modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acid include, but are not limited to, nucleic acid sulfur derivatives and thiophosphate derivatives, and those resistant to degradation of polynucleoside amides and oligonucleoside amides. The antisense nucleic acid of the present invention can be preferably designed according to the following policy. That is, to make the antisense nucleic acid more stable in the cell, to increase the cell permeability of the antisense nucleic acid, to increase the affinity for the target sense strand, and to be antitoxic if toxic Make sense nucleic acids less toxic.
Thus, many modifications are known in the art, such as J. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247, 1992; Vol. 8, pp. 395, 1992; ST Crooke et al. Ed ., Antisense Research and Applications, CRC Press, 1993, etc.
The antisense nucleic acid of the present invention may be altered or contain modified sugars, bases and bonds, provided in special forms such as liposomes, microspheres, applied by gene therapy, It can be given in an added form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( For example, hydrophobic substances such as phospholipids and cholesterols). Preferred lipids to be added include cholesterol and derivatives thereof (for example, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 ′ end or 5 ′ end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid, which prevents degradation by a nuclease such as exonuclease or RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
The inhibitory activity of the antisense nucleic acid can be examined using the transformant of the present invention, the in vivo or in vitro gene expression system of the present invention, or the in vivo or in vitro translation system of the protein of the present invention. The nucleic acid can be applied to cells by various known methods.
[0037]
The protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the protein of the present invention), the DNA encoding the protein or partial peptide of the present invention (hereinafter abbreviated as the DNA of the present invention) An antibody against the protein or partial peptide of the present invention or a salt thereof (hereinafter sometimes abbreviated as the antibody of the present invention), and an antisense nucleotide of the DNA of the present invention (hereinafter referred to as the antisense nucleotide of the present invention). The application of which may be abbreviated) will be described.
[0038]
Since the expression of the protein of the present invention increases in cancer tissues, it can be used as a disease marker. That is, it is useful as a marker for early diagnosis in cancer tissues, determination of the severity of symptoms, and prediction of disease progression. Therefore, a medicament containing an antisense nucleotide of a gene encoding the protein of the present invention, a compound or a salt thereof that inhibits the activity of the protein of the present invention, or an antibody against the protein of the present invention is, for example, colon cancer, breast cancer, lung cancer. , Prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer It can be used as a prophylactic / therapeutic agent for cancer such as brain tumor or blood tumor, apoptosis inducer and the like.
[0039]
(1) Screening of drug candidate compounds for disease
Since the expression of the protein of the present invention increases in cancer tissue and has an inhibitory effect on apoptosis, a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention is, for example, colon cancer, Breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, It can be used as a prophylactic / therapeutic agent for cancer such as pancreatic cancer, brain tumor or blood tumor. It can also be used as an apoptosis action regulator, preferably an apoptosis inducer.
In addition, since the protein of the present invention has an activity of suppressing apoptosis induced by an anticancer agent (eg, paclitaxel, etc.), a compound that regulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention The salt can also be used as an anticancer drug sensitivity enhancer.
Therefore, the protein of the present invention is useful as a reagent for screening a compound or a salt thereof that modulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention.
That is, the present invention relates to a compound that modulates (promotes or inhibits, preferably inhibits) the activity (eg, ligand binding activity, signal transduction activity, etc.) of the protein of the present invention, characterized by using the protein of the present invention A salt screening method is provided.
More specifically, for example, (i) a ligand-binding activity or signaling activity of a cell having the ability to produce the protein of the present invention, and (ii) a cell having the ability to produce the protein of the present invention and a test compound A method for screening a compound or a salt thereof that modulates (promotes or inhibits, preferably inhibits) the activity of the protein of the present invention, which is characterized by comparing the ligand binding activity or signal transduction activity of the mixture, is used.
In the above screening method, for example, in the case of (i) and (ii), ligand binding activity or signal transduction activity is described in a known method, for example, J. Immunol. 166 (1), pages 15-19, 2001. Measure and compare according to the method of or similar to the above.
Specifically, when screening for a compound having a ligand binding promotion or intracellular signal transduction promoting action, (i) a reporter containing the protein expression vector of the present invention in a promoter or enhancer containing a binding sequence of NF-κB. When introduced into a cell together with the gene and cultured, (ii) the protein expression vector of the present invention is introduced into the cell together with a reporter gene containing the binding sequence of NF-κB in a promoter or enhancer, and in the presence of the test compound The reporter expression level when cultured is compared. The reporter expression level can be measured by using reporter protein activity using luciferase activity, alkaline phosphatase activity, or the like as an index.
Specifically, when screening for a compound having a ligand binding inhibition or intracellular signal transduction inhibition action, (i) a reporter containing the protein expression vector of the present invention in a promoter or enhancer containing a binding sequence of NF-κB. Introduced into the cell together with the gene, and if necessary, (a) a microbial cell disruption solution, a microorganism culture supernatant, a eukaryotic cell disruption solution, a eukaryotic cell culture supernatant, or a solution containing a ligand, (b) the ligand itself or ( c) when cultured with a substance having a binding activity equivalent to that of a natural ligand; and (ii) the protein expression vector of the present invention together with a reporter gene containing the binding sequence of NF-κB in a promoter or enhancer. Introduce into cells, and if necessary, (a) microbial cell disruption fluid, microbial culture supernatant, eukaryotic cell disruption fluid, eukaryotic cell culture supernatant Ligand-containing solution, (b) ligand itself or (c) a substance having binding activity equivalent to that of a natural ligand is added, and the amount of reporter expression when cultured in the presence of a test compound is measured and compared. To do.
Examples of the ligand include bacterial-derived lipopeptides (preferably triacylated lipopeptides), or N-palmitoyl-S-dipalmitoylglyceryl (Pam _Three ) Cys- Ser- (Lys) _Four (CSK _Four ), N-palmitolyl-S-dilaurylgryceryl (N-Pam-S-Lau ₂ ) CSK _Four Triacyl peptides such as Borrelia burgdorferi and outer-surface lipoprotein (OspA) are used.
The reporter expression level can be measured by using reporter protein activity using luciferase activity, alkaline phosphatase activity, or the like as an index.
In addition, the signal transduction activity can be measured by measuring and comparing the apoptotic activity against anticancer agents (eg, paclitaxel, etc.) in the presence or absence of expression of proteins (eg, TLR1 etc.) used in the present invention. . Apoptotic activity against an anticancer drug (eg, paclitaxel, etc.) in the presence and absence of a ligand can be measured and compared by a known method to measure signal transduction activity.
Furthermore, in order to measure the signal transduction of TLR1 used in the present invention with higher sensitivity, a cell having the ability to express a TLR1 signal-related molecule (eg, TLR2, MD2, etc.) may be used. The above activity may be measured by forcibly expressing a TLR1 signal-related molecule (eg, TLR2, MD2, etc.).
As the cell having the ability to produce the protein of the present invention, for example, a host (transformant) transformed with a vector containing the above-described DNA encoding the protein of the present invention is used. As the host, animal cells such as COS7 cells, CHO cells, HEK293 cells are preferably used. For the screening, for example, a transformant in which the protein of the present invention is expressed on the cell membrane by culturing by the aforementioned method is preferably used. The method for culturing cells capable of expressing the protein of the present invention is the same as the method for culturing the transformant of the present invention described above.
Examples of the test compound include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like.
For example, a test compound in which the ligand binding activity or signal transduction activity in the case of (ii) is reduced by about 20% or more, preferably 30% or more, more preferably about 50% or more compared to the case of (i) above. As a compound that inhibits the activity of the protein of the present invention, the ligand binding activity or signal transduction activity in the case of (ii) above is about 20% or more, preferably 30% or more, more preferably compared to the case of (i) above. Can be selected as a compound that promotes the activity of the protein of the present invention.
[0040]
The compound having the activity of inhibiting the activity of the protein of the present invention is a safe and low-toxic drug for suppressing the physiological activity of the protein of the present invention, such as colon cancer, breast cancer, lung cancer, prostate cancer, and esophagus. Cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, etc. It is useful as a prophylactic / therapeutic agent for cancer, apoptosis inducer, anticancer agent sensitivity enhancer and the like.
The compound having the activity of promoting the activity of the protein of the present invention is useful as a safe and low toxic pharmaceutical for enhancing the action of the protein of the present invention.
The compound or salt thereof obtained using the screening method or screening kit of the present invention is, for example, a peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract, animal tissue extract. Or a compound selected from plasma and the like. As the salt of the compound, the same salts as those of the peptide of the present invention described above can be used.
[0041]
Furthermore, since the gene encoding the protein of the present invention is also increased in expression in cancer tissue, a compound or a salt thereof that regulates the expression of the gene encoding the protein of the present invention may be used, for example, breast cancer, colon cancer, breast cancer, Lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreas It is useful as a prophylactic / therapeutic agent for cancer such as brain tumor or blood tumor, apoptosis inducer, anticancer agent sensitivity enhancer and the like.
Therefore, the DNA of the present invention is useful as a reagent for screening for a compound or a salt thereof that regulates (promotes or inhibits, preferably inhibits) the expression of a gene encoding the protein of the present invention.
As a screening method, (iii) when cells having the ability to produce the protein of the present invention were cultured, and (iv) cells having the ability to produce the protein used in the present invention were cultured in the presence of the test compound. A screening method characterized by comparison with the case.
In the above method, the expression level of the gene (specifically, the amount of the protein of the present invention or the amount of mRNA encoding the protein) in the cases of (iii) and (iv) is measured and compared.
Examples of the cell having the ability to produce the test compound and the protein of the present invention include those described above.
The amount of protein is measured by a known method, for example, by using an antibody that recognizes the protein of the present invention, the protein present in a cell extract or the like according to a method such as Western analysis, ELISA method or the like or a method equivalent thereto. can do.
The amount of mRNA is measured by a known method, for example, Northern hybridization using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a probe, or PCR using a nucleic acid containing SEQ ID NO: 2 or a part thereof as a primer. It can be measured according to the method or a method according to it.
For example, a test compound that inhibits the gene expression level in the case of (iv) by about 20% or more, preferably 30% or more, more preferably about 50% or more compared to the case of (iii). As a compound that suppresses the expression of the gene encoding the protein of (iv), the gene expression level in the case of (iv) is about 20% or more, preferably 30% or more, more preferably compared to the case of (iii) above. A test compound that increases by about 50% or more can be selected as a compound that promotes the expression of the gene encoding the protein of the present invention.
[0042]
The screening kit of the present invention contains cells having the ability to produce the protein or partial peptide used in the present invention or a salt thereof, or the protein or partial peptide used in the present invention.
The compound or salt thereof obtained using the screening method or screening kit of the present invention is the above-mentioned test compound, for example, peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, plant extract. A compound selected from animal tissue extracts, plasma, and the like, or a salt thereof, and a compound or a salt thereof that modulates the activity (eg, ligand binding activity or signal transduction activity) of the protein of the present invention.
As the salt of the compound, the same salt as the protein salt of the present invention described above can be used.
A compound that modulates (preferably inhibits) the activity of the protein of the present invention or a salt thereof, and a compound that modulates (preferably inhibits) the expression of a gene encoding the protein of the present invention or a salt thereof are, for example, colon cancer, Breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, It is useful as a prophylactic / therapeutic agent for cancer such as pancreatic cancer, brain tumor or blood tumor, apoptosis inducer, anticancer agent sensitivity enhancer and the like.
When the compound or salt thereof obtained using the screening method or screening kit of the present invention is used as the above-mentioned pharmaceutical, it can be formulated according to conventional means.
[0043]
For example, compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules). Syrup, emulsion, suspension and the like. Such a composition is produced by a known method and contains a carrier, diluent or excipient usually used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
As a composition for parenteral administration, for example, injections, suppositories and the like are used, and injections are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, intravenous injections, intraarticular injections. Includes dosage forms such as agents. Such an injection is prepared according to a known method, for example, by dissolving, suspending or emulsifying the antibody or a salt thereof in a sterile aqueous or oily liquid usually used for injection. As an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. A suppository used for rectal administration is prepared by mixing the above-mentioned compound or a salt thereof with an ordinary suppository base.
[0044]
The above oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), suppositories, etc., and usually 5 to 500 mg, especially 5 to 100 mg for injections, Other dosage forms preferably contain 10 to 250 mg of the above antibody.
Each of the above-described compositions may contain other active ingredients as long as undesirable interactions are not caused by blending with the above compounds.
Since the preparation thus obtained is safe and has low toxicity, for example, humans or warm-blooded animals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, birds, cats, dogs, monkeys, chimpanzees, etc. ) Orally or parenterally.
The dose of the compound or a salt thereof varies depending on its action, target disease, administration subject, administration route, etc. For example, a compound or salt thereof that modulates the activity of the protein of the present invention for the purpose of treating breast cancer is used. When administered orally, generally for adults (with a body weight of 60 kg), the compound or salt thereof is about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-0.1 per day. 20 mg is administered. When administered parenterally, the single dose of the compound or a salt thereof varies depending on the administration subject, target disease, etc., but for example, a compound that modulates the activity of the protein of the present invention for the purpose of treating breast cancer or When the salt is usually administered to an adult (in a body weight of 60 kg) in the form of an injection, the compound or a salt thereof is about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 1 day. It is convenient to administer about 0.1 to 10 mg to a cancer lesion site by injection. In the case of other animals, an amount converted per 60 kg body weight can be administered.
Moreover, the said compound can be used in combination with the existing anticancer agent (for example, paclitaxel, docetaxel etc.), and the side effect which damages the normal cell of an anticancer agent is reduced. In this case, the administration time is not limited, and these may be administered simultaneously to the administration subject or may be administered with a time difference. The dose can be appropriately selected based on the clinically used dose. Moreover, the compounding ratio of the said compound and anticancer agent can be suitably selected according to an administration subject, an administration route, an object disease, a symptom, a combination, etc.
[0045]
(2) Quantification of the protein of the present invention, its partial peptide or its salt
Since the antibody against the protein of the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) can specifically recognize the protein of the present invention, the quantification of the protein of the present invention in the test solution, particularly It can be used for quantification by sandwich immunoassay.
That is, the present invention
(I) The antibody of the present invention is competitively reacted with a test solution and the labeled protein of the present invention, and the ratio of the labeled protein of the present invention bound to the antibody is measured. A method for quantifying the protein of the present invention in a test solution, and
(Ii) The test solution and the antibody of the present invention insolubilized on the carrier and the labeled another antibody of the present invention are reacted simultaneously or successively, and then the activity of the labeling agent on the insolubilized carrier is measured. The method for quantifying the protein of the present invention in a test solution is provided.
In the above quantification method (ii), it is desirable that one antibody is an antibody that recognizes the N-terminal part of the protein of the present invention and the other antibody is an antibody that reacts with the C-terminal part of the protein of the present invention.
[0046]
In addition to quantification of the protein of the present invention using a monoclonal antibody against the protein of the present invention (hereinafter sometimes referred to as the monoclonal antibody of the present invention), detection by tissue staining or the like can also be performed. For these purposes, the antibody molecule itself may be used, or the F (ab ′) of the antibody molecule. ₂ , Fab ′, or Fab fractions may be used.
The method for quantifying the protein of the present invention using the antibody of the present invention is not particularly limited, and an antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (for example, the amount of protein) in the solution to be measured is not limited. Any measurement method may be used as long as it is a measurement method in which the amount is detected by chemical or physical means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used, but the sandwich method described later is particularly preferable in view of sensitivity and specificity.
Examples of the labeling agent used in the measurement method using the labeling substance include radioisotopes, enzymes, fluorescent substances, luminescent substances, lanthanide elements, and the like. Examples of radioisotopes include [ ¹²⁵ I], [ ¹³¹ I], [ ^Three H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin-avidin system can also be used for binding of an antibody or antigen and a labeling agent.
[0047]
For insolubilization of an antigen or antibody, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize or immobilize a protein or an enzyme may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
In the sandwich method, the test solution is reacted with the insolubilized monoclonal antibody of the present invention (primary reaction), and further labeled with another monoclonal antibody of the present invention (secondary reaction), and then on the insolubilized carrier. By measuring the activity of the labeling agent, the amount of the protein of the present invention in the test solution can be quantified. The primary reaction and the secondary reaction may be performed in the reverse order, or may be performed simultaneously or at different times. The labeling agent and the insolubilization method can be the same as those described above. In the immunoassay by the sandwich method, the antibody used for the solid phase antibody or the labeling antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
In the method for measuring the protein of the present invention by the sandwich method of the present invention, the monoclonal antibody of the present invention used for the primary reaction and the secondary reaction is preferably an antibody having a different site to which the protein of the present invention binds. That is, the antibody used in the primary reaction and the secondary reaction, for example, when the antibody used in the secondary reaction recognizes the C-terminus of the protein of the present invention, the antibody used in the primary reaction is preferably For example, an antibody that recognizes the N end other than the C end is used.
[0048]
The monoclonal antibody of the present invention can be used in measurement systems other than the sandwich method, for example, competitive method, immunometric method, nephrometry, and the like.
In the competition method, the antigen in the test solution and the labeled antigen are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody are separated. (B / F separation), the labeling amount of either B or F is measured, and the amount of antigen in the test solution is quantified. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol, a liquid phase method using a second antibody against the antibody, and a solid-phased antibody is used as the first antibody, or As the first antibody, a soluble method is used, and a solid phase method using a solid phase antibody as the second antibody is used.
In the immunometric method, the antigen in the test solution and the immobilized antigen are subjected to a competitive reaction with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the antigen in the test solution is separated. Are reacted with an excess amount of labeled antibody, and then a solid phased antigen is added to bind the unreacted labeled antibody to the solid phase, and then the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the test solution.
In nephrometry, the amount of insoluble precipitate produced as a result of the antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of antigen in the test solution is small and only a small amount of precipitate is obtained.
[0049]
In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of the protein of this invention, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequential Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. “Enzyme Immunoassay” (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid. )) (From Academic Press ) And the like can be referred to.
As described above, the protein of the present invention can be quantified with high sensitivity by using the antibody of the present invention.
Furthermore, when an increase (or decrease) in the concentration of the protein of the present invention is detected by quantifying the concentration of the protein of the present invention using the antibody of the present invention, for example, colorectal cancer, breast cancer, lung cancer, prostate Cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor Alternatively, it can be diagnosed that the cancer is a blood tumor or is likely to be affected in the future. In addition, when the concentration of the protein of the present invention is high, it can be diagnosed that the effect of administering an anticancer agent (eg, paclitaxel, etc.) is low.
The antibody of the present invention can be used for detecting the protein of the present invention present in a subject such as a body fluid or tissue. In addition, for the production of an antibody column used for purifying the protein of the present invention, detection of the protein of the present invention in each fraction during purification, analysis of the behavior of the protein of the present invention in a test cell, etc. Can be used.
[0050]
(3) Genetic diagnostic agents
The DNA of the present invention can be used, for example, as a probe for humans or warm-blooded animals (eg, rats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) Can detect an abnormality (gene abnormality) of DNA or mRNA encoding the protein of the present invention or a partial peptide thereof, for example, damage, mutation or decreased expression of the DNA or mRNA, It is useful as a gene diagnostic agent for increase or overexpression.
The gene diagnosis using the DNA of the present invention includes, for example, the known Northern hybridization and PCR-SSCP method (Genomics, Vol. 5, pp. 874-879 (1989), Proceedings of the -National Academy of Sciences of USA (Proceedings of the National Academy of Sciences of the United States of America), 86, 2766-2770 (1989)).
For example, when overexpression or decrease is detected by Northern hybridization, or when DNA mutation is detected by PCR-SSCP method, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer Cancer such as liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor Can be diagnosed as likely.
[0051]
(4) Medicine containing antisense nucleotide
The antisense nucleotide of the present invention, which can complementarily bind to the DNA of the present invention and suppress the expression of the DNA, has low toxicity, and functions of the protein of the present invention or the DNA of the present invention in vivo (eg, Ligand binding activity or signal transduction activity), for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, gastric cancer, liver cancer, biliary tract cancer, spleen cancer, renal cancer. , Bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, preventive and therapeutic agent for cancer such as brain tumor or blood tumor, apoptosis inducer, anticancer agent sensitivity enhancer Can be used as such.
When the antisense nucleotide is used as the above-mentioned preventive / therapeutic agent, it can be formulated and administered according to a known method.
Further, for example, after inserting the above-mentioned antisense nucleotide alone or into an appropriate vector such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector, etc., in accordance with conventional means, a human or mammal (eg, rat, rabbit) , Sheep, pigs, cattle, cats, dogs, monkeys, etc.) orally or parenterally. The antisense nucleotide can be formulated as it is or with a physiologically recognized carrier such as an adjuvant for promoting intake, and can be administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving cellular uptake efficiency, the above-mentioned antisense nucleotides are formulated alone (injection) together with carriers such as liposomes, and are intravenously, subcutaneously, intraarticularly. Alternatively, it may be administered to a cancerous lesion or the like.
The dosage of the antisense nucleotide varies depending on the target disease, administration subject, administration route, etc. For example, when the antisense nucleotide of the present invention is administered for the purpose of treating breast cancer, it is generally used for adults (weight 60 kg). ), About 0.1 to 100 mg of the antisense nucleotide is administered per day.
Furthermore, the antisense nucleotide can also be used as a diagnostic oligonucleotide probe for examining the presence and expression status of the DNA of the present invention in tissues and cells.
Similarly to the above-mentioned antisense nucleotide, double-stranded RNA containing a part of RNA encoding the protein of the present invention, ribozyme containing a part of RNA encoding the protein of the present invention, and the like are also included in the gene of the present invention. Since the expression can be suppressed and the function of the protein used in the present invention or the DNA used in the present invention in vivo can be suppressed, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophagus Cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, etc. It can be used as a prophylactic / therapeutic agent for cancer, apoptosis inducer, anticancer agent sensitivity enhancer and the like.
The double-stranded RNA can be produced by designing based on the sequence of the polynucleotide of the present invention according to a known method (eg, Nature, 411, 494, 2001).
The ribozyme can be designed and produced based on the sequence of the polynucleotide of the present invention according to a known method (eg, TRENDS in Molecular Medicine, 7, 221, 2001). For example, it can be produced by linking a known ribozyme to a part of the RNA encoding the protein of the present invention. Examples of the RNA encoding the protein of the present invention include a portion (RNA fragment) adjacent to the cleavage site on the RNA of the present invention that can be cleaved by a known ribozyme.
When the above double-stranded RNA or ribozyme is used as the prophylactic / therapeutic agent, it can be formulated and administered in the same manner as the antisense polynucleotide.
Similar to the above-described antisense nucleotide, the protein of the present invention or the aptamer of the DNA encoding the protein of the present invention can also suppress the expression of the gene of the present invention, and the protein used in the present invention or the present invention in vivo. Can suppress the function of DNA used in, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, Prevention / treatment agent for bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, apoptosis inducer, anticancer agent sensitivity enhancer, etc. Can be used as
Aptamers can be produced according to known methods.
[0052]
(5) Medicament containing the antibody of the present invention
The antibody of the present invention having the action of neutralizing the activity of the protein of the present invention includes, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, Kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, preventive or therapeutic agent for cancer such as brain tumor or blood tumor, apoptosis inducer, anticancer agent It can be used as a safe drug with low toxicity such as a sensitivity enhancer.
The prophylactic / therapeutic agent for the above-mentioned diseases containing the antibody of the present invention has low toxicity and can be used as a solution or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig). , Cattle, cats, dogs, monkeys, etc.) or or parenterally (eg, intra-articular administration). The dose varies depending on the administration subject, target disease, symptom, administration route, etc. For example, when used for treatment or prevention of breast cancer in adults, the dose of the antibody of the present invention is usually 0. About 0.01-20 mg / kg body weight, preferably about 0.1-10 mg / kg body weight, more preferably about 0.1-5 mg / kg body weight, about 1-5 times a day, preferably 1-3 times a day It is convenient to administer by a powder inhaler about once. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the administration comprises the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as dosage forms suitable for oral or parenteral administration (eg, intravenous administration).
Each of the above-described compositions may contain other active ingredients as long as they do not cause an unfavorable interaction by blending with the antibody.
[0053]
(6) "A cancer preventive / therapeutic agent comprising a compound having a Toll-like receptor activity-modulating action or a salt thereof" and "a compound having a Toll-like receptor expression-controlling action or a salt thereof of the present invention About the preventive / therapeutic agent for cancer comprising
The “compound having a Toll-like receptor activity-modulating action” may be any compound as long as it has a Toll-like receptor activity-regulating action, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer. , Stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, etc. It is used as a prophylactic / therapeutic agent for cancer, apoptosis inducer, anticancer drug sensitivity enhancer, etc.
The “compound having a Toll-like receptor expression regulating action” may be any compound as long as it has a Toll-like receptor expression regulating action, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer. , Stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor, etc. It is used as a prophylactic / therapeutic agent for cancer, apoptosis inducer, anticancer drug sensitivity enhancer, etc.
The prophylactic / therapeutic agent is produced in the same manner as described above.
[0054]
(7) DNA transfer animals
The present invention includes a DNA encoding a foreign protein of the present invention (hereinafter abbreviated as the foreign DNA of the present invention) or a mutant DNA thereof (sometimes abbreviated as the foreign mutant DNA of the present invention). A human mammal is provided.
That is, the present invention
(1) a non-human mammal having the exogenous DNA of the present invention or a mutant DNA thereof,
(2) the animal according to (1), wherein the non-human mammal is a rodent;
(3) The animal according to (2), wherein the rodent is a mouse or a rat, and
(4) Provided is a recombinant vector containing the exogenous DNA of the present invention or a mutant DNA thereof and capable of being expressed in mammals.
A non-human mammal having an exogenous DNA of the present invention or a mutant DNA thereof (hereinafter abbreviated as a DNA-transferred animal of the present invention) can be used for embryos including unfertilized eggs, fertilized eggs, spermatozoa and primordial cells thereof. Preferably, at the stage of embryonic development in non-human mammal development (more preferably at the single cell or fertilized egg cell stage and generally prior to the 8-cell stage), the calcium phosphate method, electric pulse method, lipofection method, aggregation method, It can be produced by transferring the target DNA by the microinjection method, particle gun method, DEAE-dextran method or the like. Further, by the DNA transfer method, the target exogenous DNA of the present invention can be transferred to somatic cells, living organs, tissue cells, etc., and can be used for cell culture, tissue culture, etc. The DNA-transferred animal of the present invention can also be produced by fusing the above-mentioned embryo cells with a known cell fusion method.
Examples of non-human mammals include cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like. Among them, rodents, especially mice (for example, C57BL / 6 strain, DBA2 strain, etc. as pure strains) are relatively short in terms of ontogeny and biological cycle from the viewpoint of creating a disease animal model system, and are easy to reproduce. As a system, B6C3F ₁ System, BDF ₁ System, B6D2F ₁ Strain, BALB / c strain, ICR strain, etc.) or rat (for example, Wistar, SD, etc.) is preferred.
Examples of the “mammal” in the recombinant vector that can be expressed in the mammal include humans and the like in addition to the above non-human mammals.
[0055]
The exogenous DNA of the present invention is not the DNA of the present invention inherently possessed by a non-human mammal but the DNA of the present invention once isolated and extracted from the mammal.
Examples of the mutant DNA of the present invention include those in which a mutation (for example, mutation or the like) has occurred in the base sequence of the original DNA of the present invention, specifically, addition of a base, deletion, substitution to another base, etc. The resulting DNA is used, and abnormal DNA is also included.
The abnormal DNA means DNA that expresses the abnormal protein of the present invention, and for example, DNA that expresses a protein that suppresses the function of the normal protein of the present invention is used.
The exogenous DNA of the present invention may be derived from mammals of the same or different species as the target animal. In transferring the DNA of the present invention to a target animal, it is generally advantageous to use the DNA as a DNA construct bound downstream of a promoter that can be expressed in animal cells. For example, when transferring the human DNA of the present invention, the expression of DNA derived from various mammals (for example, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) having the DNA of the present invention having high homology with this. The DNA of the present invention is highly expressed by microinjecting a DNA construct (eg, a vector) conjugated with the human DNA of the present invention downstream of various promoters into a fertilized egg of a target mammal, for example, a mouse fertilized egg. DNA transfer mammals can be created.
[0056]
Examples of the expression vector for the protein of the present invention include plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis, plasmids derived from yeast, bacteriophages such as λ phage, retroviruses such as Moloney leukemia virus, animal viruses such as vaccinia virus and baculovirus. Etc. are used. Of these, plasmids derived from Escherichia coli, plasmids derived from Bacillus subtilis or plasmids derived from yeast are preferably used.
Examples of promoters that regulate the above DNA expression include: (1) promoters of DNA derived from viruses (eg, simian virus, cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus, poliovirus, etc.); ▼ Promoters from various mammals (human, rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), such as albumin, insulin II, uroplakin II, elastase, erythropoietin, endothelin, muscle creatine kinase, glial fibrillary acidity Protein, glutathione S-transferase, platelet-derived growth factor β, keratin K1, K10 and K14, collagen type I and type II, cyclic AMP-dependent protein kinase βI subunit, dyst Lophine, tartrate-resistant alkaline phosphatase, atrial natriuretic factor, endothelial receptor tyrosine kinase (generally abbreviated as Tie2), sodium potassium adenosine 3 kinase (Na, K-ATPase), neurofilament light chain, metallothionein I IIA, metalloproteinase 1 tissue inhibitor, MHC class I antigen (H-2L), H-ras, renin, dopamine β-hydroxylase, thyroid peroxidase (TPO), peptide chain elongation factor 1α (EF-1α), β actin , Α and β myosin heavy chain, myosin light chain 1 and 2, myelin basic protein, thyroglobulin, Thy-1, immunoglobulin, heavy chain variable region (VNP), serum amyloid P component, myoglobin, troponin C, smooth muscle α A Chin, pre-pro-enkephalin A, such as a promoter, such as vasopressin is used. Among these, a cytomegalovirus promoter capable of being highly expressed throughout the body, a human peptide chain elongation factor 1α (EF-1α) promoter, human and chicken β-actin promoters, and the like are preferable.
The vector preferably has a sequence (generally called a terminator) that terminates transcription of the target messenger RNA in a DNA-transferred mammal. For example, the sequence of each DNA derived from viruses and various mammals The SV40 terminator of simian virus or the like is preferably used.
[0057]
In addition, the splicing signal of each DNA, enhancer region, part of intron of eukaryotic DNA, etc. 5 ′ upstream of the promoter region, between the promoter region and the translation region, or the translation region for the purpose of further expressing the target foreign DNA It is also possible to connect it 3 'downstream of the target.
The normal translation region of the protein of the present invention includes liver, kidney, thyroid cell, fibroblast-derived DNA derived from humans or various mammals (eg, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, etc.) and commercially available DNA. As a raw material, complementary DNA prepared by a known method as a whole or a part of genomic DNA or from RNA derived from liver, kidney, thyroid cells, or fibroblasts can be obtained. Further, exogenous abnormal DNA can produce a translation region obtained by mutating a normal protein translation region obtained from the cells or tissues described above by a point mutagenesis method.
The translation region can be prepared as a DNA construct that can be expressed in a metastasized animal by an ordinary DNA engineering technique in which it is linked downstream of the promoter and optionally upstream of the transcription termination site.
The transfer of the foreign DNA of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the subject mammal. The presence of the foreign DNA of the present invention in the embryo cells of the produced animal after DNA transfer means that all the progeny of the produced animal retain the foreign DNA of the present invention in all the germ cells and somatic cells. means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the foreign DNA of the present invention in all of its germ cells and somatic cells.
The non-human mammal to which the exogenous normal DNA of the present invention has been transferred can be subcultured in a normal breeding environment as a DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. .
The transfer of the exogenous DNA of the present invention at the fertilized egg cell stage is ensured to be excessively present in all germ cells and somatic cells of the target mammal. Excessive exogenous DNA of the present invention is present in the embryo cells of the produced animal after the DNA transfer. This means that all the offspring of the produced animal have the exogenous DNA of the present invention in all the germ cells and somatic cells. Means. The offspring of this type of animal that has inherited the foreign DNA of the present invention has an excess of the foreign DNA of the present invention in all germ cells and somatic cells.
By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA in excess.
[0058]
In the non-human mammal having the normal DNA of the present invention, the normal DNA of the present invention is highly expressed, and finally the function of the endogenous normal DNA is promoted, thereby finally causing the hyperfunction of the protein of the present invention. It can develop and can be used as a disease model animal. For example, the normal DNA-transferred animal of the present invention can be used to elucidate the hyperfunction of the protein of the present invention, the pathological mechanism of the disease associated with the protein of the present invention, and the examination of treatment methods for these diseases. Is possible.
Further, since the mammal to which the exogenous normal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, a preventive / therapeutic agent for diseases related to the protein of the present invention, such as colon cancer, Breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, It can also be used in screening tests for prophylactic / therapeutic agents for cancer such as pancreatic cancer, brain tumor or blood tumor.
On the other hand, the non-human mammal having the exogenous abnormal DNA of the present invention can be subcultured in a normal breeding environment as the DNA-bearing animal after confirming that the exogenous DNA is stably retained by mating. Furthermore, the target foreign DNA can be incorporated into the aforementioned plasmid and used as a raw material. A DNA construct with a promoter can be prepared by a normal DNA engineering technique. The abnormal DNA transfer of the present invention at the fertilized egg cell stage is ensured to be present in all germ cells and somatic cells of the target mammal. The presence of the abnormal DNA of the present invention in the embryo cell of the produced animal after DNA transfer means that all the offspring of the produced animal have the abnormal DNA of the present invention in all the germ cells and somatic cells. The offspring of this type of animal that has inherited the foreign DNA of the present invention has the abnormal DNA of the present invention in all of its germ cells and somatic cells. By obtaining a homozygous animal having the introduced DNA in both homologous chromosomes and mating the male and female animals, all the offspring can be bred and passaged so as to have the DNA.
[0059]
In the non-human mammal having the abnormal DNA of the present invention, the abnormal DNA of the present invention is highly expressed, and finally the functional inactive form of the protein of the present invention is inhibited by inhibiting the function of the endogenous normal DNA. It may become refractory and can be used as a model animal for the disease state. For example, by using the abnormal DNA-transferred animal of the present invention, it is possible to elucidate the pathological mechanism of the functional inactive refractory of the protein of the present invention and to examine a method for treating this disease.
Further, as a specific applicability, the abnormal DNA high-expressing animal of the present invention exhibits the function inhibition (dominant negative action) of the normal protein by the abnormal protein of the present invention in the functional inactive refractory disease of the protein of the present invention. It becomes a model to be solved.
Further, since the mammal to which the foreign abnormal DNA of the present invention has been transferred has an increased symptom of the released protein of the present invention, a preventive / therapeutic agent for the protein of the present invention or a functional inactive refractory disease such as the large intestine Cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid gland It can also be used in screening tests for prophylactic / therapeutic agents for cancer such as cancer, pancreatic cancer, brain tumor or blood tumor.
Further, as other applicability of the above-mentioned two kinds of DNA transfer animals of the present invention, for example,
(1) Use as a cell source for tissue culture,
(2) Peptides specifically expressed or activated by the protein of the present invention by directly analyzing DNA or RNA in the tissue of the DNA-transferred animal of the present invention or by analyzing peptide tissue expressed by the DNA Analysis of the relationship with
(3) Cell of tissue having DNA is cultured by a standard tissue culture technique, and using these, research on the function of cells from tissue generally difficult to cultivate,
(4) Screening for drugs that enhance cell function by using the cells described in (3) above, and
(5) Isolation and purification of the mutant protein of the present invention and production of its antibody are conceivable.
[0060]
Furthermore, using the DNA-transferred animal of the present invention, clinical symptoms of diseases related to the protein of the present invention, including functional inactive refractories of the protein of the present invention, can be examined, and the protein of the present invention More detailed pathological findings in each organ of the disease model related to the disease can be obtained, and it is possible to contribute to the development of a new treatment method and further to the study and treatment of secondary diseases caused by the disease.
Moreover, each organ can be taken out from the DNA-transferred animal of the present invention, and after minced, it is possible to obtain free DNA-transferred cells, culture them, or systematize the cultured cells with a proteolytic enzyme such as trypsin. . Furthermore, the protein of the present invention and its action can be investigated by identifying the protein-producing cells of the present invention, examining the relationship with apoptosis, differentiation or proliferation, or the signal transduction mechanism in them, and examining their abnormalities. It becomes an effective research material for.
Furthermore, in order to develop a therapeutic agent for the diseases related to the protein of the present invention, including the functional inactive refractory of the protein of the present invention, using the DNA-transferred animal of the present invention, the above-described test method and quantification It is possible to provide an effective and rapid screening method for a therapeutic agent for the disease using a method or the like. Moreover, it is possible to examine and develop a DNA therapy for a disease associated with the protein of the present invention using the DNA-transferred animal of the present invention or the exogenous DNA expression vector of the present invention.
[0061]
(8) Knockout animals
The present invention provides a non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated and the DNA expression-deficient non-human mammal of the present invention.
That is, the present invention
(1) a non-human mammalian embryonic stem cell in which the DNA of the present invention is inactivated,
(2) The embryonic stem cell according to (1), wherein the DNA is inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli),
(3) The embryonic stem cell according to (1), which is neomycin-resistant,
(4) The embryonic stem cell according to item (1), wherein the non-human mammal is a rodent.
(5) The embryonic stem cell according to (4), wherein the rodent is a mouse,
(6) the DNA expression-deficient non-human mammal in which the DNA of the present invention is inactivated,
(7) The DNA can be inactivated by introducing a reporter gene (eg, β-galactosidase gene derived from E. coli), and the reporter gene can be expressed under the control of a promoter for the DNA of the present invention (6) The non-human mammal according to Item,
(8) The non-human mammal according to (6), wherein the non-human mammal is a rodent.
(9) The non-human mammal according to item (8), wherein the rodent is a mouse, and
(10) A screening method for a compound or a salt thereof that promotes or inhibits promoter activity against the DNA of the present invention, which comprises administering a test compound to the animal according to item (7) and detecting the expression of a reporter gene I will provide a.
[0062]
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated refers to suppressing the DNA expression ability by artificially adding mutation to the DNA of the present invention possessed by the non-human mammal, or By substantially losing the activity of the protein of the present invention encoded by the DNA, the DNA has substantially no ability to express the protein of the present invention (hereinafter sometimes referred to as the knockout DNA of the present invention). ) Non-human mammal embryonic stem cells (hereinafter abbreviated as ES cells).
As the non-human mammal, the same ones as described above are used.
As a method for artificially adding a mutation to the DNA of the present invention, for example, a part or all of the DNA sequence can be deleted or another DNA can be inserted or replaced by a genetic engineering technique. For example, the knockout DNA of the present invention may be prepared by shifting the codon reading frame or destroying the function of the promoter or exon by these mutations.
[0063]
Specific examples of non-human mammalian embryonic stem cells in which the DNA of the present invention is inactivated (hereinafter abbreviated as the DNA inactivated ES cell of the present invention or the knockout ES cell of the present invention) A DNA of the present invention possessed by a non-human mammal is isolated, and a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, lacZ (β-galactosidase gene), cat (chloramphenicol acetyl) Inserting a reporter gene or the like typified by a transferase gene) to destroy the function of the exon, or inserting a DNA sequence (for example, a polyA addition signal) that terminates the transcription of the gene into an intron between exons, Preventing the synthesis of complete messenger RNA As a result, a DNA strand having a DNA sequence constructed so as to destroy the gene (hereinafter abbreviated as a targeting vector) is introduced into the chromosome of the animal by, for example, homologous recombination, and the obtained ES cell Southern hybridization analysis using the DNA sequence of the present invention or the vicinity thereof as a probe, or PCR method using the DNA sequence of the targeting vector and the DNA sequence of the neighboring region other than the DNA of the present invention used for the preparation of the targeting vector as primers And can be obtained by selecting the knockout ES cell of the present invention.
In addition, as the original ES cell for inactivating the DNA of the present invention by homologous recombination method or the like, for example, those already established as described above may be used, or in accordance with the known Evans and Kaufman method. Or newly established. For example, in the case of mouse ES cells, currently 129 ES cells are generally used, but since the immunological background is unclear, an alternative purely immunologically genetic background For example, for the purpose of obtaining ES cells with clear clarification, for example, B57 improved by reducing the number of eggs collected in C57BL / 6 mice and C57BL / 6 by crossing with DBA / 2 ₁ Mouse (F of C57BL / 6 and DBA / 2 ₁ ) Can be used well. BDF ₁ In addition to the advantage that the number of eggs collected is high and the eggs are strong, the mouse has C57BL / 6 mice as a background. Therefore, when ES cells obtained using these mice are used to produce pathological model mice, C57BL / 6 mice can be advantageously used in that the genetic background can be changed to C57BL / 6 mice by backcrossing.
When establishing ES cells, blastocysts on the third day after fertilization are generally used, but in addition to this, many blastocysts can be efficiently obtained by collecting eggs and culturing them to blastocysts. Early embryos can be obtained.
Although both male and female ES cells may be used, male ES cells are usually more convenient for producing germline chimeras. In addition, it is desirable to discriminate between males and females as soon as possible in order to reduce troublesome culture work.
[0064]
One example of a method for determining the sex of an ES cell is a method of amplifying and detecting a gene in the sex-determining region on the Y chromosome by PCR. If this method is used, it is conventionally about 10 times for karyotype analysis. ⁶ In contrast to the number of cells required, the number of ES cells of about 1 colony (about 50) is sufficient, so primary selection of ES cells in the early stage of culture can be performed by male / female discrimination. Yes, the early labor of the culture can be greatly reduced by enabling the selection of male cells at an early stage.
The secondary selection can be performed, for example, by confirming the number of chromosomes by the G-banding method. The number of chromosomes of the obtained ES cell is preferably 100% of the normal number. However, if it is difficult due to physical operations during establishment, the gene of the ES cell is knocked out, and then normal cells (for example, chromosomes in mice) It is desirable to clone again into cells where the number is 2n = 40.
The embryonic stem cell line obtained in this way is usually very proliferative, but it tends to lose its ontogenic ability, so it needs to be subcultured carefully. For example, in a carbon dioxide incubator (preferably 5% carbon dioxide, 95% air or 5% oxygen, 5%) in the presence of LIF (1-10000 U / ml) on suitable feeder cells such as STO fibroblasts. Culturing is carried out by a method such as culturing at about 37 ° C. with carbon dioxide gas, 90% air, and at the time of passage, for example, a trypsin / EDTA solution (usually 0.001-0.5% trypsin / 0.1-5 mM EDTA, Preferably, the cells are converted into single cells by treatment with about 0.1% trypsin / 1 mM EDTA and seeded on newly prepared feeder cells. Such passage is usually carried out every 1-3 days. At this time, it is desirable to observe the cells, and when morphologically abnormal cells are found, the cultured cells should be discarded.
ES cells can be differentiated into various types of cells such as parietal muscle, visceral muscle, and myocardium by monolayer culture to high density or suspension culture until a cell conglomerate is formed under appropriate conditions. [MJ Evans and MH Kaufman, Nature 292, 154, 1981; GR Martin Proceedings of National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA) 78, 7634, 1981; TC Doetschman et al., Journal of Embrology and Experimental Morphology, 87, 27, 1985], ES cells of the present invention. The DNA expression-deficient cells of the present invention obtained by differentiation are useful for in vitro cell biology of the protein of the present invention.
[0065]
The DNA expression deficient non-human mammal of the present invention can be distinguished from normal animals by measuring the mRNA level of the animal using a known method and comparing the expression level indirectly.
As the non-human mammal, those similar to the above can be used.
The DNA expression-deficient non-human mammal of the present invention is a DNA in which, for example, a targeting vector prepared as described above is introduced into mouse embryonic stem cells or mouse egg cells, and the DNA of the targeting vector of the present invention is inactivated by the introduction. The DNA of the present invention can be knocked out by homologous recombination in which the sequence replaces the DNA of the present invention on the chromosome of mouse embryonic stem cells or mouse egg cells by gene homologous recombination.
A cell in which the DNA of the present invention is knocked out is obtained by analyzing the DNA sequence of the Southern hybridization analysis or targeting vector using the DNA sequence of or near the DNA of the present invention as a probe and the mouse used for the targeting vector of the present invention. It can be determined by analysis by a PCR method using a DNA sequence in a neighboring region other than DNA as a primer. When a non-human mammalian embryonic stem cell is used, a cell line in which the DNA of the present invention is inactivated by gene homologous recombination is cloned, and the cell is placed at a suitable time, for example, an 8-cell non-human mammal. It is injected into animal embryos or blastocysts, and the produced chimeric embryos are transplanted into the uterus of the non-human mammal that is pseudopregnant. The produced animal is a chimeric animal composed of both cells having the normal DNA locus of the present invention and cells having the artificially mutated DNA locus of the present invention. When some of the germ cells of the chimeric animal have the mutated DNA locus of the present invention, all tissues are artificially mutated from the population obtained by mating such a chimeric individual with a normal individual. It can be obtained by selecting an individual composed of the added cells having the DNA locus of the present invention, for example, by determining the coat color. The individuals thus obtained are usually individuals with deficient hetero-expression of the protein of the present invention, and individuals with deficient hetero-expression of the protein of the present invention are crossed and homozygous expression of the protein of the present invention from their offspring. Failure individuals can be obtained.
[0066]
When using an egg cell, for example, a transgenic non-human mammal having a targeting vector introduced into the chromosome can be obtained by injecting a DNA solution into the nucleus of the egg cell by a microinjection method. Compared to mammals, it can be obtained by selecting those having a mutation at the DNA locus of the present invention by gene homologous recombination.
Thus, an individual in which the DNA of the present invention is knocked out can be reared in a normal breeding environment after confirming that the individual animal obtained by mating has also been knocked out.
In addition, germline acquisition and retention may be followed in accordance with conventional methods. That is, a homozygous animal having the inactivated DNA in both homologous chromosomes can be obtained by mating male and female animals possessed by the inactivated DNA. The obtained homozygous animal can be efficiently obtained by rearing the mother animal in a state where there are 1 normal individual and multiple homozygous animals. By breeding male and female heterozygotes, homozygotes and heterozygotes having the inactivated DNA are bred and passaged.
The non-human mammal embryonic stem cell in which the DNA of the present invention is inactivated is very useful for producing the non-human mammal deficient in DNA expression of the present invention.
In addition, since the non-human mammal deficient in DNA expression of the present invention lacks various biological activities that can be induced by the protein of the present invention, it is a model for diseases caused by inactivation of the biological activity of the protein of the present invention. Therefore, it is useful for investigating the causes of these diseases and examining treatment methods.
[0067]
(8a) Screening method for compounds having therapeutic / preventive effects on diseases caused by deficiency or damage of DNA of the present invention
The DNA expression-deficient non-human mammal of the present invention can be used for screening a compound having a therapeutic / preventive effect on a disease caused by the deficiency or damage of the DNA of the present invention.
That is, the present invention is caused by the deficiency or damage of the DNA of the present invention, which comprises administering a test compound to the non-human mammal deficient in DNA expression of the present invention and observing and measuring changes in the animal. Provided is a screening method for a compound having a therapeutic / preventive effect on a disease such as cancer, or a salt thereof.
Examples of the non-human mammal deficient in DNA expression of the present invention used in the screening method include those described above.
Examples of the test compound include peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, etc. These compounds are novel compounds. It may be a known compound.
Specifically, the non-human mammal deficient in DNA expression of the present invention is treated with a test compound, compared with an untreated control animal, and tested using changes in each organ, tissue, disease symptom, etc. of the animal as an index. The therapeutic / prophylactic effect of the compound can be tested.
As a method for treating a test animal with a test compound, for example, oral administration, intravenous injection and the like are used, and can be appropriately selected according to the symptoms of the test animal, the properties of the test compound, and the like. The dosage of the test compound can be appropriately selected according to the administration method, the properties of the test compound, and the like.
[0068]
For example, colorectal cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer When screening a compound having a therapeutic / preventive effect against cancer such as thyroid cancer, pancreatic cancer, brain tumor or blood tumor, the test compound is administered to the non-human mammal deficient in DNA expression of the present invention and tested. Differences in the degree of onset of cancer and the degree of cancer healing are observed over time in the above tissues with no compound administration group.
In the screening method, when a test compound is administered to a test animal, the test compound is improved when the disease symptoms of the test animal are improved by about 10% or more, preferably about 30% or more, more preferably about 50% or more. It can be selected as a compound having a therapeutic / prophylactic effect on the above-mentioned diseases.
The compound obtained by using the screening method is a compound selected from the test compounds described above, and has a therapeutic / prophylactic effect on diseases caused by deficiency or damage of the protein of the present invention. It can be used as a medicine such as a safe and low-toxic preventive / therapeutic agent. Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids, organic acids, etc.) and bases (eg, alkali metals, etc.). And the like, and physiologically acceptable acid addition salts are particularly preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
A medicament containing the compound obtained by the screening method or a salt thereof can be produced in the same manner as the aforementioned medicament containing the protein of the present invention.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, administration subject, administration route, etc. For example, when the compound is administered orally, generally in an adult (weight 60 kg) breast cancer patient, The compound is administered at about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, target disease, etc., but the compound is usually administered to an adult (60 kg) breast cancer patient in the form of an injection. In this case, it is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg of the compound per day by intravenous injection. In the case of other animals, an amount converted per 60 kg can be administered.
[0069]
(8b) Screening method for compounds that promote or inhibit the activity of the promoter for the DNA of the present invention
The present invention provides a compound that promotes or inhibits the activity of a promoter for the DNA of the present invention, which comprises administering a test compound to a non-human mammal deficient in DNA expression of the present invention and detecting the expression of a reporter gene, or a compound thereof A salt screening method is provided.
In the screening method described above, the DNA expression-deficient non-human mammal of the present invention is inactivated by introducing a reporter gene among the DNA expression-deficient non-human mammals of the present invention described above, A gene capable of expressing the reporter gene under the control of a promoter for the DNA of the present invention is used.
Examples of the test compound are the same as described above.
As the reporter gene, the same ones as described above are used, and a β-galactosidase gene (lacZ), a soluble alkaline phosphatase gene, a luciferase gene, or the like is preferable.
In the non-human mammal of the present invention in which the DNA of the present invention is replaced with a reporter gene, the expression of the substance encoded by the reporter gene is traced because the reporter gene exists under the control of the promoter for the DNA of the present invention. By doing so, the activity of the promoter can be detected.
For example, when a part of the DNA region encoding the protein of the present invention is replaced with the β-galactosidase gene (lacZ) derived from Escherichia coli, the protein of the present invention is originally replaced with the tissue expressing the protein of the present invention. Β-galactosidase is expressed. Therefore, for example, by staining with a reagent that is a substrate of β-galactosidase such as 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal), the present invention can be easily performed. The expression state of the protein in the animal can be observed. Specifically, the protein-deficient mouse of the present invention or a tissue section thereof is fixed with glutaraldehyde or the like, washed with phosphate buffered saline (PBS), and then stained with X-gal at room temperature or around 37 ° C. Then, after reacting for about 30 minutes to 1 hour, the tissue specimen is washed with a 1 mM EDTA / PBS solution to stop the β-galactosidase reaction and observe the coloration. Moreover, you may detect mRNA which codes lacZ according to a conventional method.
The compound obtained by using the screening method or a salt thereof is a compound selected from the above-described test compounds, and is a compound that promotes or inhibits the promoter activity for the DNA of the present invention.
The compound obtained by the screening method may form a salt. Examples of the salt of the compound include physiologically acceptable acids (eg, inorganic acids) and bases (eg, organic acids). In particular, physiologically acceptable acid addition salts are preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
[0070]
Since the compound or its salt that promotes or inhibits the promoter activity for the DNA of the present invention can regulate the expression of the protein of the present invention and the function of the protein, for example, colon cancer, breast cancer, lung cancer, prostate Cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or It is useful as a prophylactic / therapeutic agent for cancers such as blood tumors.
Furthermore, compounds derived from the compounds obtained by the above screening can be used as well.
The pharmaceutical containing the compound or salt thereof obtained by the screening method can be produced in the same manner as the pharmaceutical containing the protein of the present invention or salt thereof.
Since the preparation thus obtained is safe and low toxic, it can be used, for example, in humans or mammals (eg, rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, etc.). Can be administered.
The dose of the compound or a salt thereof varies depending on the target disease, administration subject, administration route, and the like. For example, when a compound that inhibits the promoter activity against the DNA of the present invention is orally administered, generally an adult (body weight) In a breast cancer patient (as 60 kg), the compound is administered at about 0.1-100 mg, preferably about 1.0-50 mg, more preferably about 1.0-20 mg per day. When administered parenterally, the single dose of the compound varies depending on the administration subject, the target disease, etc. For example, a compound that inhibits the promoter activity against the DNA of the present invention is usually administered in the form of an injection (in adults) When administered to a breast cancer patient (as 60 kg), about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg is administered by intravenous injection per day. It is convenient to do. In the case of other animals, an amount converted per 60 kg can be administered.
Thus, the non-human mammal deficient in DNA expression of the present invention is extremely useful in screening for compounds or salts thereof that promote or inhibit the activity of the promoter for the DNA of the present invention. It can greatly contribute to the investigation of the causes of various diseases caused or the development of preventive / therapeutic agents.
In addition, using DNA containing the promoter region of the protein of the present invention, genes encoding various proteins are ligated downstream thereof, and this is injected into an egg cell of an animal to produce a so-called transgenic animal (gene transgenic animal). Once created, the protein can be specifically synthesized and its action in the living body can be studied. Furthermore, if a suitable reporter gene is bound to the above promoter portion and a cell line that expresses it is established, a low molecular weight compound that specifically promotes or suppresses the production ability of the protein itself of the present invention in the body. Can be used as a search system.
[0071]
In this specification, when a base, an amino acid, or the like is indicated by an abbreviation, it is based on an abbreviation by the IUPAC-IUB Commission on Biochemical Nomenclature or a common abbreviation in the field, and examples thereof are described below. In addition, when there are optical isomers with respect to amino acids, L form is shown unless otherwise specified.

[0072]
In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.

[0073]
The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
The amino acid sequence of TLR1 is shown.
[SEQ ID NO: 2]
This shows the base sequence of DNA encoding TLR1 having the amino acid sequence represented by SEQ ID NO: 1.
[SEQ ID NO: 3]
The base sequence of DNA containing the full-length gene encoding TLR1 is shown.
[SEQ ID NO: 4]
The base sequence of the antisense used in Example 2 is shown.
[SEQ ID NO: 5]
The base sequence of the primer used in Example 2 is shown.
[SEQ ID NO: 6]
The base sequence of the primer used in Example 2 is shown.
[SEQ ID NO: 7]
The base sequence of the primer used in Example 2 is shown.
[SEQ ID NO: 8]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 9]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 10]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 11]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 12]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 13]
The base sequence of the primer used in Example 3 is shown.
[SEQ ID NO: 14]
The base sequence of the DNA fragment inserted in the vector in Example 3 is shown.
[0074]
In the following, the present invention will be more specifically described with reference to examples, but the present invention is not limited thereto.
【Example】
Example 1
In order to clarify the genes that are specifically upregulated in breast cancer tissue, total RNA (Table 1) extracted from 2 cases of breast cancer tissue and 23 other normal tissues was used as a material, oligonucleotide microarray (Human Genome U95A , U95B, U95C, U95D, U95E; Affymetrix)).
The experimental method was in accordance with the Affymetrix experimental analysis technical manual. As a result, expression of the TLR1 gene was detected only in breast cancer tissues. All normal tissues analyzed were below the detection limit (Table 2).
[0075]
[Table 1]

[Table 2]

[0076]
Example 2
In order to analyze the effect of TLR1 gene, whose expression was specifically observed on breast cancer, on apoptosis, a TLR1 antisense oligonucleotide introduction experiment was performed. First, after designing an antisense (SEQ ID NO: 4) to the nucleotide sequence represented by SEQ ID NO: 3, a phosphorothioated oligonucleotide was synthesized, purified by HPLC, and used in an introduction experiment (Amersham Pharmacia Biotech). As a control oligonucleotide, the reverse sequence (SEQ ID NO: 5) of the base sequence shown in SEQ ID NO: 4 was similarly phosphorothioated and used after HPLC purification (Amersham Pharmacia Biotech).
Breast cancer cell line MDA-MB-231 (In Vitro, 14 (11), 911-915, 1978, purchased from ATCC) was used as a test cell, and 18 × 10 8 the day before oligonucleotide introduction. ^Three Cells were seeded in a 24-well plate (Falcon). OligofectAMINE (manufactured by Invitrogen) was used for oligonucleotide introduction, and the protocol was followed. Seven hours after introduction, RNA was extracted according to the protocol of RNeasy mini kit (manufactured by Qiagen), and cDNA was prepared using ThermoScriptRT-PCR system (manufactured by GIBCO BRL). Two primers (SEQ ID NO: 6 and SEQ ID NO: 7), 18S rRNA primers and competitors from QuantumRNA 18S Internal Standards (manufactured by Ambion) were added simultaneously according to the manual for RCR reaction, and multiplex PCR was performed. After the PCR reaction, the reaction solution was electrophoresed, and the intensity of the band stained with ethidium bromide was corrected with the band intensity of 18S rRNA using BioImageIQ software (manufactured by Genomic Solutions), and then compared.
On the other hand, the effect on apoptosis was compared with the control oligo-introduced sample using Cell death detection ELISA (manufactured by Roche) on the third day after introduction of the antisense oligonucleotide.
As a result, the expression (RNA) of TLR1 decreased to 55% 7 hours after the introduction of the antisense, compared to the time when the control (reverse) oligonucleotide was introduced (100%). On the third day after introduction, apoptosis increased to 186% compared to control (100%).
From this, it was revealed that TLR1 is not only expressed specifically in cancer but also involved in proliferation and apoptosis of cancer cells.
[0077]
Example 3
(1) Preparation of TLR1 and MD2 expression vectors
In order to prepare a TLR1 expression system, the full-length gene of TLR1 was cloned.
Two types of primers (SEQ ID NO: 8 and SEQ ID NO: 9) were used, and Marathon ready cDNA library (Clontech) was used as a template to perform PCR according to the protocol attached to Pfu polymerase (Straragene). PCR was performed using 20 μl of a reaction solution containing 2 μl of 10 × Pfu buffer, 0.4 μl of 10 mM each dNTP mixture (manufactured by Clontech), 0.8 μl of each of the two primers prepared in 20 mM, 0.5 μl of template cDNA solution, and 0.1 μl of Pfu polymerase. After the pretreatment at 94 ° C. for 2 minutes, the reaction was carried out for 42 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 3 minutes and then 72 ° C. for 7 minutes.
After the PCR reaction, the products were separated by agarose gel electrophoresis, and the target band was cut out and cloned into pCR4Blunt-TOPO vector (Invitrogen). As a result of confirming the base sequence of the inserted fragment, it was consistent with the human TLR1 base sequence, so PCR was performed using the vector obtained above as a template using two additional primers (SEQ ID NO: 10 and SEQ ID NO: 11). went. PCR was carried out using 20 μl of a reaction solution containing 2 μl of 10 × Pfu buffer, 0.4 μl of 10 mM each dNTP mixture (manufactured by Clontech), 0.2 μl of each of the two primers prepared in 50 mM, 0.5 μl of template cDNA solution, and 0.4 μl of Pfu polymerase. After the pretreatment at 94 ° C for 2 minutes, the reaction was conducted at 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 3 minutes, followed by 25 cycles, followed by 72 ° C for 7 minutes. After PCR reaction using Pfu polymerase (Straragene), A was added to both ends of the PCR product according to the protocol attached to pCR4-TOPO vector (Invitrogen), and the products were separated by agarose gel electrophoresis. The band was cut out and cloned into pCR4-TOPO vector (Invitrogen). After confirming that there are no errors in the sequence, digest with restriction enzyme BclII (Nippon Gene) and insert into p3XFLAG-CMV-14 (SIGMA) treated with BclII using Ligation kit ver.2 (Takara) did. It was confirmed again that there was no error in the sequence, and the obtained TLR1 expression vector was used in the following experiment.
On the other hand, PCR was performed using KOD polymerase (manufactured by Toyobo) using two types of primers (SEQ ID NO: 12 and SEQ ID NO: 13) and a human spleen-derived cDNA library (manufactured by Clontech) as a template. PCR was performed using 20 μl of reaction solution containing 2 μl of 10 × KOD buffer, 1.6 μl of 2.5 mM each dNTP mixture (manufactured by Clontech), 0.2 μl of each of the above two primers prepared to 50 mM, 1 μl of template cDNA solution, 0.2 μl of KOD polymerase, After the pretreatment at 94 ° C. for 2 minutes, the reaction was carried out for 42 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 3 minutes and then 72 ° C. for 7 minutes.
After the PCR reaction, the product was separated by agarose gel electrophoresis, the target band was excised, digested with restriction enzymes KpnI and NotI, and then digested with the same restriction enzymes into pcDNA3.1 / Hyg (+) (manufactured by Invitrogen) Ligation was performed to obtain an MD2 expression vector. The base sequence of the inserted fragment into this vector is shown in SEQ ID NO: 14. As a result of confirming the base sequence of the inserted fragment, it was consistent with the base sequence of human MD2.
[0078]
(2) Effect of TLR1 on reporter activity
Next, in order to see the activity of TLR1 in cancer cells, the expression vector obtained in (1) above was introduced into NCI-H1299 cells (purchased from ATCC) to observe the effect on various reporter activities.
The day before transfection of the expression vector shown below, NCI-H1299 strain was 2 × 10 ^Four Each was seeded in a 24-well plate (manufactured by Falcon) and transfected using Fugene6 (manufactured by Roche) according to the attached protocol. The TLR1 expression vector obtained in Example 3 (1) above and the pcDNA3.1 vector used as a control were used at 0.19 μg / well. As a reporter, 0.19 μg / well of a vector having AP1 site or NF-κB site upstream of the secreted alkaline phosphatase gene (both manufactured by Clontech) or a vector having an ISRE site upstream of the firefly luciferase gene (manufactured by Clontech), The pRL-TK (manufactured by Toyo Ink) used for correcting the reporter activity was 0.13 μg / well. The reporter secreted alkaline phosphatase activity or firefly luciferase activity was measured on the second day after transfection using the Great EscAPe SEAP Fluorescence Detection Kit kit (manufactured by Clontech) and the Picker Gene Dual Sea Pansy Double Luciferase Assay System (manufactured by Toyo Ink). Measured according to Sea shiitake luciferase activity expressed by pRL-TK was measured according to the protocol of Picker Gene Dual Sea Pansy Double Luciferase Assay System (manufactured by Toyo Ink), and the reporter activity value was corrected.
As a result, TLR1 expression increased AP1 reporter activity by 5.6 times, NF-κB reporter activity by 3.3 times, and ISRE reporter activity by 1.7 times compared to the pcDNA3.1 vector used as a control.
From this, it was found that by expressing TLR1, AP1 signal, NF-κB signal, and ISRE signal are activated in the lung cancer-derived NCI-H1299 strain without any additional ligand.
Therefore, by performing these promoter-reporter assays, it is possible to screen for compounds having a TLR1 signal regulating action.
[0079]
(3) Effects of paclitaxel on apoptosis induction
Next, in order to see the effect of TLR1 signal operation on cancer, the effect on apoptosis induction by paclitaxel was observed.
5 x 10 HCT116 (purchased from ATCC) the day before expression vector transfection ^Five Individual 10 cm dishes (manufactured by Falcon) were seeded, and transfection was performed according to the protocol of Fugene 6 (manufactured by Roche). Combinations shown in Table 3 so that the TLR1 expression vector and MD2 expression vector obtained in Example 3 (1) above or the pcDNA3.1 vector (manufactured by Invitrogen) used as a control were each 11 μg per dish. Used in. Cells were harvested by trypsinization 4 hours after transfection and 3 × 10 ^Three N-palmitoyl-S-dilauryl-glyceryl CSK, which is a TLR1 ligand, is seeded individually in a 96-well plate (Falcon). _Four (Pam _Three CSK _Four ) (Manufactured by Peptide Institute) (1 μg / ml) and / or paclitaxel (manufactured by Sigma), an anticancer agent, was added (0.125 μM) to observe the effect on apoptosis.
After 2 days of culture in a 96-well plate, apoptosis was detected using Cell Death Detection ELISA plus kit (Roche) according to the attached protocol. As shown in FIG. 1, when paclitaxel was present, the absorbance was higher than when it was absent, and it was confirmed that apoptosis was induced by paclitaxel (white bar vs. gray bar). In contrast, Pam _Three CSK _Four Since the absorbance decreased due to the addition of, it was found that apoptosis was decreased by the addition of TLR1 ligand (gray bar vs. hatched bar). Pam _Three CSK _Four It was found that the apoptosis induced by paclitaxel was reduced by transfection of TLR1 and MD2 without adding (grey bar vs. black bar).
From this, it was revealed that TLR1 signal has an effect of attenuating apoptosis by an anticancer agent.
[0080]
【The invention's effect】
The protein used in the present invention is a cancer diagnostic marker, and therefore, a compound or a salt thereof that regulates (preferably inhibits) the activity of the protein, or a compound that regulates (preferably inhibits) gene expression of the protein Or a salt thereof, an antibody of the present invention, an antisense nucleotide of the present invention, for example, colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney As a safe drug for cancer, bladder cancer, uterine cancer, ovarian cancer, testicular cancer, thyroid cancer, pancreatic cancer, cancer such as brain tumor or blood tumor, and pro-apoptotic agent It can be used and can also be used as a therapeutic agent for cancer that is resistant to anticancer agents. In addition, in order to increase sensitivity to anticancer agents (eg, paclitaxel, etc.), the dose of existing anticancer agents can be reduced to reduce side effects.
[0081]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the results of examining the effect of paclitaxel on apoptosis induction.

Claims (12)

SEQ ID NO: containing a complementary base sequence or a portion thereof to the base sequence of DNA encoding a protein having an amino acid sequence represented by 1, comprising an antisense nucleotide to inhibit translation of the protein Cancer preventive / therapeutic agent. SEQ ID NO: amino acid sequence or SEQ ID NO: represented by 1: In the amino acid sequence represented by 1, met protein several amino acid 1 is deleted, added, containing insertions and / or substituted amino acid sequence A preventive / therapeutic agent for cancer comprising an antibody against a protein having a intracellular signaling activity of Toll-like receptor or a salt thereof.   Cancer is colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, The preventive / therapeutic agent according to claim 1 or 2, which is testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor. SEQ ID NO: amino acid sequence or SEQ ID NO: represented by 1: In the amino acid sequence represented by 1, met protein several amino acid 1 is deleted, added, containing insertions and / or substituted amino acid sequence A diagnostic agent for cancer comprising an antibody against a protein having a intracellular signaling activity of Toll-like receptor or a salt thereof. SEQ ID NO: amino acid sequence or SEQ ID NO: represented by 1: In the amino acid sequence represented by 1, several amino acid 1 is deleted, added, a protein having an insertion, and / or substituted amino acid sequence A diagnostic agent for cancer comprising DNA encoding a protein having intracellular signaling activity of Toll-like receptor .   Cancer is colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, ovarian cancer, The diagnostic agent according to claim 4 or 5, which is testicular cancer, thyroid cancer, pancreatic cancer, brain tumor or blood tumor. A protein containing an amino acid sequence that is identical to the amino acid sequence represented by SEQ ID NO: 1 or in which one to several amino acids are deleted, added, inserted and / or substituted in the amino acid sequence represented by SEQ ID NO: 1. A method for screening a prophylactic / therapeutic agent for cancer, comprising using a protein having a intracellular signaling activity of Toll-like receptor or a salt thereof. SEQ ID NO: amino acid sequence or SEQ ID NO: represented by 1: In the amino acid sequence represented by 1, met protein several amino acid 1 is deleted, added, containing insertions and / or substituted amino acid sequence A screening kit for a prophylactic / therapeutic agent for cancer, comprising a protein having a intracellular signaling activity of Toll-like receptor or a salt thereof. SEQ ID NO: amino acid sequence or SEQ ID NO: represented by 1: In the amino acid sequence represented by 1, several amino acid 1 is deleted, added, a protein having an insertion, and / or substituted amino acid sequence A screening method for a prophylactic / therapeutic agent for cancer, which comprises using a DNA encoding a protein having intracellular signaling activity of a Toll-like receptor . SEQ ID NO: amino acid sequence or SEQ ID NO: represented by 1: In the amino acid sequence represented by 1, several amino acid 1 is deleted, added, a protein having an insertion, and / or substituted amino acid sequence A screening kit for a prophylactic / therapeutic agent for cancer, comprising DNA encoding a protein having intracellular signaling activity of Toll-like receptor . SEQ ID NO: of the amino acid sequence or SEQ ID NO 1: In the amino acid sequence represented by 1, or several amino acids are deleted. 1, additionally, a protein containing insertions and / or substituted amino acid sequence A method for screening an apoptosis-inducing agent, comprising using a protein having a intracellular signaling activity of a Toll-like receptor or a salt thereof. SEQ ID NO: 1 amino acid sequence or SEQ ID NO represented: In the amino acid sequence represented by 1, met several amino acids are deleted. 1, addition, proteins with insertions and / or substituted amino acid sequence A method for screening an apoptosis-inducing agent, comprising using a DNA encoding a protein having intracellular signaling activity of a Toll-like receptor .

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