JP2001128677A - Promoter regulating expression of adam10 - Google Patents
Promoter regulating expression of adam10Info
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Abstract
Description
【発明の属する技術分野】本発明は、脊椎動物、特にヒ
トの組織に広く分布するADAM10遺伝子の転写調節領域
（プロモーター領域）及びその利用法に関する。The present invention relates to a transcriptional regulatory region (promoter region) of the ADAM10 gene widely distributed in vertebrate tissues, particularly human tissues, and a method of using the same.
【従来の技術】ADAM10は細胞表面に存在するディスイン
テグリンとメタロプロテアーゼドメインを有するADAMフ
ァミリーに属するタンパク質の一つである。もともとは
Drosophiaの発生過程に関わるプロテアーゼとしてKuzba
nian（あるいはkuz）が知られていたが、そのホモログ
がADAM10であることが判明した (Curr. Biol., 7, 716
(1997))。2. Description of the Related Art ADAM10 is one of the proteins belonging to the ADAM family having a disintegrin and a metalloprotease domain present on the cell surface. Originally
Kuzba as a protease involved in the development of Drosophia
nian (or kuz) was known, but its homolog was found to be ADAM10 (Curr. Biol., 7 , 716
(1997)).
【０００１】現在までにADAM10の活性として、腫瘍壊死
因子αのプロセシング(FEBS Lett.,400, 333(1997); J.
Biol. Chem., 272, 24588 (1997))、ミエリン塩基性タ
ンパク質およびタイプIVコラーゲンの切断(Biochem.
J., 317, 45 (1996); Biochem. Biophys. Res. Commu
n., 245, 594 (1997))、アミロイド前駆タンパク質のα
セクレターゼ活性が知られている(Proc. Natl. Acad. S
ci. USA., 96, 3922 (1999))。従って、ADAM10は多発性
硬化症に代表される脱髄性疾患、腎疾患における基底膜
病変、アルツハイマー性痴呆の発症に関わっている可能
性が示唆されている。また、ADAM10はある種の腫瘍細胞
に多く発現していることが報告されており、腫瘍転移へ
の関連を示唆する報告がある(Biochem. Biophys. Res.
Commun., 235, 437 (1997))。ADAM10が変形性関節症の
患者の軟骨細胞において高発現していることが示された
(Biochem. Biophys. Res. Commun., 230, 335 (1997);
J. Histochem. Cytochem., 46, 723 (1997))。このこと
からADAM10が軟骨マトリックスコンポーネントを変性
し、変形性関節症発症へ導く可能性が示唆された。ADAM
10が様々な疾患に関与する可能性が示唆されるものの、
ADAM10の転写制御がどの様になされているかは、全く未
知であり、ADAM10のプロモーター領域の解析、調節因子
の同定、転写に影響をあたえる化合物スクリーニング系
の構築が待たれた。To date, the activity of ADAM10 has been reported as the processing of tumor necrosis factor α (FEBS Lett., 400, 333 (1997);
Biol. Chem., 272, 24588 (1997)), cleavage of myelin basic protein and type IV collagen (Biochem.
J., 317, 45 (1996); Biochem. Biophys. Res.
n., 245, 594 (1997)), α of amyloid precursor protein
Secretase activity is known (Proc. Natl. Acad. S.
ci. USA., 96 , 3922 (1999)). Therefore, it has been suggested that ADAM10 may be involved in the development of demyelinating diseases represented by multiple sclerosis, basement membrane lesions in renal diseases, and Alzheimer's dementia. Also, it has been reported that ADAM10 is highly expressed in certain types of tumor cells, and there is a report suggesting a relationship to tumor metastasis (Biochem. Biophys. Res.
Commun., 235, 437 (1997)). ADAM10 is highly expressed in chondrocytes of patients with osteoarthritis
(Biochem. Biophys. Res.Commun., 230, 335 (1997);
J. Histochem. Cytochem., 46, 723 (1997)). This suggested that ADAM10 may degenerate cartilage matrix components, leading to the development of osteoarthritis. ADAM
Although it is suggested that 10 may be involved in various diseases,
How the transcription of ADAM10 is regulated is completely unknown, and analysis of the ADAM10 promoter region, identification of regulatory factors, and construction of a compound screening system that affects transcription have been awaited.
【０００２】[0002]
【発明が解決しようとする課題】本発明の課題は、ADAM
10遺伝子の転写制御を行っている、プロモーター領域を
クローニングし、塩基配列を明らかにすると共に、ADAM
10の発現に影響を与える化合物をスクリーニングする系
を構築することにある。An object of the present invention is to provide an ADAM.
Cloning the promoter region, which controls the transcription of 10 genes, clarifying the nucleotide sequence, and
The object of the present invention is to construct a system for screening compounds that affect the expression of 10.
【０００３】[0003]
【課題を解決するための手段】本発明者らは、ADAM10遺
伝子コード領域の上流にあるプロモーター領域をインバ
ースPCRによりクローニングし、塩基配列を明らかにし
た。更に、クローニングしたADAM10遺伝子のプロモータ
ー領域の下流に分泌性アルカリフォスファターゼ（PLA
P）をつないで、ブタ軟骨細胞及びNIH3T3細胞に導入
し、ADAM10遺伝子プロモーター領域の転写活性を、アル
カリフォスファターゼの活性として測定する系を構築し
た。これにより、ADAM10の発現に影響を与える調節物質
をスクリーニングすることが可能となった。すなわち本
発明は、ADAM10遺伝子プロモーター、そのプロモーター
を含んだベクター、そのベクターを導入した細胞、その
細胞を使ったADAM10転写制御に影響を与える化合物のス
クリーニング法に関する。具体的には、 (1).配列番号１に記載の塩基配列からなるDNA。 (2).配列番号１に記載の塩基配列からなるDNAとストリ
ンジェントな条件でハイブリダイズし、ADAM10の転写調
節を行うプロモーター活性を持ったDNA。ここでストリ
ンジェントな条件の一例は、65℃ 4 x SSCにおけるハイ
ブリダイゼーション、次いで65℃で１時間0.1 X SSC中
での洗浄である。また別法としてストリンジェントな条
件は、50%ホルムアミド中42℃ 4 X SSCである。 (3).配列番号１に記載の塩基配列の一部からなるDNAを
プライマーとしてPCRにより増幅されるDNAで、ADAM10の
転写調節を行うプロモーター活性を持ったDNA。ここで
配列番号１に記載された塩基配列の一部とは、配列番号
１に記載された塩基配列中の連続した１０塩基、好まし
くは１５塩基、更に好ましくは２５塩基からなるDNAを
意味する。 (4).(1)ないし(3)に記載のDNAを含むベクター。 (5).(4)に記載のベクターを導入した細胞。 (6).(5)に記載の細胞を用いることを特徴とする、ADAM1
0遺伝子の発現調節に影響を与える化合物をスクリーニ
ングする方法。 に関する。Means for Solving the Problems The present inventors have cloned the promoter region upstream of the coding region of the ADAM10 gene by inverse PCR to elucidate the nucleotide sequence. Furthermore, a secreted alkaline phosphatase (PLA) is located downstream of the promoter region of the cloned ADAM10 gene.
P), and introduced into porcine chondrocytes and NIH3T3 cells to construct a system for measuring the transcription activity of the ADAM10 gene promoter region as alkaline phosphatase activity. As a result, it became possible to screen for a modulator that affects the expression of ADAM10. That is, the present invention relates to an ADAM10 gene promoter, a vector containing the promoter, a cell into which the vector has been introduced, and a method for screening a compound having an effect on ADAM10 transcription control using the cell. Specifically, (1) a DNA comprising the nucleotide sequence of SEQ ID NO: 1. (2) DNA having a promoter activity that hybridizes with the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and regulates ADAM10 transcription. One example of stringent conditions here is hybridization at 65 ° C. in 4 × SSC, followed by washing in 0.1 × SSC at 65 ° C. for 1 hour. Alternatively, stringent conditions are 4 × SSC at 42 ° C. in 50% formamide. (3) A DNA that is amplified by PCR using a DNA consisting of a part of the nucleotide sequence of SEQ ID NO: 1 as a primer, and has a promoter activity for regulating the transcription of ADAM10. Here, a part of the base sequence described in SEQ ID NO: 1 means a DNA consisting of 10 consecutive bases, preferably 15 bases, and more preferably 25 bases in the base sequence described in SEQ ID NO: 1. (4) A vector containing the DNA of (1) to (3). (5) A cell into which the vector according to (4) has been introduced. (6) ADAM1 characterized by using the cell according to (5).
0 A method for screening a compound that affects the regulation of gene expression. About.
【０００４】[0004]
【発明の実施の形態】以下に本発明の実施の形態につい
て1).プロモーター領域のクローニング、2).ベクタへの
挿入、3).細胞への導入、4).ADAM10の発現に影響を与え
る薬剤のスクリーニング、の順に詳細に説明する。BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention are described below: 1) cloning of a promoter region, 2) insertion into a vector, 3) introduction into a cell, 4) influence on the expression of ADAM10. This will be described in detail in the order of drug screening.
【０００５】1).プロモーター領域のクローニング プロモーター領域は、例えば配列番号２に記載のADAM10
の配列を基にプライマーを設定し、インバースPCRを行
ってクローニングできる(Genetics, 120, 621(1998))。
具体的には、ヒトgenomic DNAを様々な制限酵素で切断
した後DNA ligaseで環状化したDNAをテンプレートと
し、ADAM10遺伝子のコード領域の一部、例えば配列番号
４及び配列番号５をプライマー（5'プライマーと3'プラ
イマーを逆に使って、プライマーの外側のDNAを増幅す
る）としてPCRを行う。増幅されたDNAバンドを切り出し
て、適当なベクター例えばpT7Blue(R)T、pUC、pBlueScr
iptに挿入し、塩基配列を決定して、ADAM10遺伝子のプ
ロモーター領域を含むクローンを選択する。別法として
は、配列番号２に記載のADAM10の配列を基にプライマー
を設定し、ヒトgenomic DNAをテンプレートとしてPCRを
行って、ADAM10遺伝子のコード領域のプローブを作製
し、ヒトgenomicライブラリーより、プローブとハイブ
リダイズするクローンを選択し、選ばれたクローンの塩
基配列を決定して、ADAM10遺伝子のプロモーター領域を
含むクローンを選択してもよい。場合によっては、得ら
れたクローンをプローブとして、更に上流配列を含むク
ローンを拾い上げても良い。[0005] 1). Cloning of promoter region The promoter region is, for example, ADAM10 described in SEQ ID NO: 2.
A primer can be set based on the above sequence and cloned by performing inverse PCR (Genetics, 120 , 621 (1998)).
Specifically, DNA obtained by cleaving human genomic DNA with various restriction enzymes and then cyclizing with DNA ligase is used as a template, and a part of the coding region of the ADAM10 gene, for example, SEQ ID NO: 4 and SEQ ID NO: 5 is used as a primer (5 ′ PCR is performed using the primer and the 3 'primer in reverse, amplifying the DNA outside the primer). The amplified DNA band is cut out and an appropriate vector such as pT7Blue (R) T, pUC, pBlueScr
After insertion into ipt, the nucleotide sequence is determined, and a clone containing the promoter region of the ADAM10 gene is selected. Alternatively, a primer is set based on the sequence of ADAM10 described in SEQ ID NO: 2, PCR is performed using human genomic DNA as a template, a probe for the coding region of the ADAM10 gene is prepared, and a human genomic library is used. A clone that hybridizes with the probe is selected, the nucleotide sequence of the selected clone is determined, and a clone containing the promoter region of the ADAM10 gene may be selected. In some cases, using the obtained clone as a probe, a clone further containing an upstream sequence may be picked up.
【０００６】2).ベクターへの挿入 クローニングされたADAM10遺伝子のプロモーター領域
は、適当なレポーター遺伝子、例えば分泌性アルカリフ
ォスファターゼ（placental alkaline phosphatase, PL
AP）、クロラムフェニコールアセチルトランスフェラー
ゼ（CAT）、ルシフェラーゼ等の遺伝子の上流に挿入
し、ADAM10遺伝子のプロモーター活性を、レポーター遺
伝子産物の活性によって測定できるようベクターを構築
することができる。具体的には、クローニングされたAD
AM10遺伝子のプロモーター領域を、適当な制限酵素で切
り出し、pPLAP (Mol. Pharmacol., 49, 860(1996))、pU
C-CAT、pSV0CAT等の制限酵素部位に挿入する。適当な制
限酵素部位が無い場合は、制限酵素切断配列を持ったオ
リゴヌクレオチドをligationする、あるいはPCRで制限
酵素部位を導入する等の手段を用いて挿入することも許
される。場合によっては、遺伝子が導入された細胞を選
択する選択マーカー、例えばNeomycin耐性遺伝子等を更
に挿入しても良い。尚、以上に記載した操作は適当なマ
ニュアル、例えばSambruck, J., Fritsch,E. F., and M
aniatis, T. (1989) Molecular Cloning: A Laboratory
Manual,Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY等に記載の方法に従って行うことができ
る。2). Insertion into a Vector The promoter region of the cloned ADAM10 gene may contain a suitable reporter gene, for example, a placental alkaline phosphatase (PL).
AP), chloramphenicol acetyltransferase (CAT), luciferase, or the like, and a vector can be constructed so that the promoter activity of the ADAM10 gene can be measured by the activity of the reporter gene product. Specifically, the cloned AD
The promoter region of the AM10 gene was cut out with an appropriate restriction enzyme, and pPLAP (Mol. Pharmacol., 49 , 860 (1996)), pU
Insert into restriction sites such as C-CAT and pSV0CAT. When there is no suitable restriction enzyme site, insertion using a means such as ligation of an oligonucleotide having a restriction enzyme cleavage sequence or introduction of a restriction enzyme site by PCR is also permitted. In some cases, a selection marker for selecting cells into which the gene has been introduced, such as a Neomycin resistance gene, may be further inserted. The operations described above are performed in appropriate manuals, for example, Sambruck, J., Fritsch, EF, and M
aniatis, T. (1989) Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring
This can be performed according to the method described in Harbor, NY and the like.
【０００７】3).細胞への導入 ADAM10遺伝子のプロモーター領域及びその下流にレポー
ター遺伝子を挿入したベクターは適当な方法、例えばリ
ン酸カルシウム共沈法、DEAE-デキストラン法、エレク
トロポレーション法、リポフェクチン法等により、哺乳
類細胞に導入できるが、効率よくDNAを導入できれば如
何なる方法も許される。哺乳類細胞にベクターを導入し
た後、もしベクターに選択マーカーが挿入されていれ
ば、それに対応した薬剤の存在下で培養し、導入した遺
伝子を持った細胞を選択する。 4).ADAM10の発現に影響を与える薬剤のスクリーニング ADAM10遺伝子のプロモーター領域及びその下流にレポー
ター遺伝子を挿入したベクターを導入した細胞を、スク
リーニングすべき化合物の存在下で培養する。適当な期
間培養した後、挿入したレポーター遺伝子の遺伝子産
物、例えばPLAP、CAT、ルシフェラーゼの活性を測定す
る。また、同時にスクリーニングすべき化合物の細胞毒
性の評価、例えばMTT法による細胞毒性の評価を行って
も良い。挿入したレポーター遺伝子の遺伝子産物の活性
を上昇させる、あるいは下降させる化合物を選択するこ
とで、ADAM10の発現に影響を与える薬剤のスクリーニン
グを行うことができる。[0007] 3). Introduction into cells A vector having a promoter region of the ADAM10 gene and a reporter gene inserted downstream thereof is prepared by an appropriate method, for example, calcium phosphate coprecipitation method, DEAE-dextran method, electroporation method, lipofectin method and the like. Although it can be introduced into mammalian cells, any method is permissible if DNA can be introduced efficiently. After the introduction of the vector into the mammalian cells, if a selection marker has been inserted into the vector, the cells are cultured in the presence of a corresponding drug, and cells having the introduced gene are selected. 4). Screening of Drugs Affecting ADAM10 Expression Cells into which a vector into which a reporter gene has been inserted downstream of the promoter region of ADAM10 gene and downstream thereof are cultured in the presence of the compound to be screened. After culturing for an appropriate period, the activity of a gene product of the inserted reporter gene, for example, PLAP, CAT, luciferase is measured. At the same time, the cytotoxicity of the compound to be screened may be evaluated, for example, the cytotoxicity may be evaluated by the MTT method. By selecting a compound that increases or decreases the activity of the gene product of the inserted reporter gene, a drug that affects the expression of ADAM10 can be screened.
【０００８】[0008]
【発明の効果】ADAM10遺伝子の上流にあるプロモーター
領域の塩基配列を解明し、またADAM10遺伝子のプロモー
ター領域、及びその一部を欠失したプロモーター領域の
下流にレポーター遺伝子をつないだベクターを作製し、
哺乳類細胞に導入して、ADAM10の発現に影響を与える化
合物をスクリーニングすることが可能となった。EFFECT OF THE INVENTION The base sequence of the promoter region upstream of the ADAM10 gene is elucidated, and a vector in which a reporter gene is linked downstream of the promoter region of the ADAM10 gene and a promoter region in which a part thereof is deleted is prepared.
It has become possible to screen for compounds that affect ADAM10 expression when introduced into mammalian cells.
【０００９】[0009]
【実施例】以下の実施例を挙げて本発明をより具体的に
説明するが、これらは本発明の範囲を制限するものでは
ない。The present invention will be described more specifically with reference to the following examples, which do not limit the scope of the present invention.
【００１０】［実施例１］インバースPCRによるヒトADA
M10フラグメントの増幅、シーケンシングおよびサブク
ローニング ヒトADAM10発現調節領域をクローニングする目的でイン
バースPCRを行った(Genetics, 120, 621(1998))。ヒト
ゲノムDNA（ノバジェン社）をBamHI, HindIII,EcoRI及
びEcoRVで一晩消化し、T4 DNA ligaseにて一晩処理し環
状DNAとしてテンプレートとした。ヒトADAM10の cDNA配
列（配列番号２）を基に配列番号４及び配列番号５のプ
ライマーを合成しPCRを行った。Hind IIIで消化したラ
イブラリーよりPCRにて得られた約2 kbのDNA断片を、0.
8％アガロースゲルを用いて電気泳動し、目的とするDNA
断片を確認した後、キアクイックゲル抽出キット（キア
ジェン社）にて回収・精製した。該DNA断片はそのままp
T7Blue(R)Tベクター（ノバジェン社）へligationした。
その後、大腸菌DH5αに導入して得られたアンピシリン
耐性株より目的のプラスミドDNAを選択・単離した。ク
ローン化された該DNA断片の塩基配列について、種々の
合成オリゴDNAをプライマーとして塩基配列を決定し、A
DAM10のcDNA配列（配列番号２）と比較した結果、得ら
れたDNA断片が１番目のエクソンの5'上流領域を含むこ
とが明らかとなった。また、ADAM10のmRNAの転写開始点
を、5' RACE解析により同定した結果、翻訳開始コドンA
TGより、239塩基上流であることが明らかとなった。転
写開始点付近にTATAボックスは見出されなかった。GCボ
ックスは翻訳開始点から-500と-509の間に位置してい
た。ADAM10の5'上流領域には５つのAP2、２つのSp1結合
サイト様の配列が見つかった。[Example 1] Human ADA by inverse PCR
Amplification, Sequencing and Subcloning of M10 Fragment Inverse PCR was performed for the purpose of cloning the human ADAM10 expression regulatory region (Genetics, 120 , 621 (1998)). Human genomic DNA (Novagen) was digested with BamHI, HindIII, EcoRI, and EcoRV overnight, treated with T4 DNA ligase overnight, and used as a template as a circular DNA. Based on the cDNA sequence of human ADAM10 (SEQ ID NO: 2), primers of SEQ ID NO: 4 and SEQ ID NO: 5 were synthesized and subjected to PCR. Approximately 2 kb of DNA fragment obtained by PCR from the library digested with Hind III was
Electrophoresis using 8% agarose gel
After confirming the fragment, it was recovered and purified using Qiaquick Gel Extraction Kit (Qiagen). The DNA fragment is p
Ligation was performed on T7Blue (R) T vector (Novagen).
Thereafter, a target plasmid DNA was selected and isolated from an ampicillin-resistant strain obtained by introduction into Escherichia coli DH5α. With respect to the nucleotide sequence of the cloned DNA fragment, the nucleotide sequence was determined using various synthetic oligo DNAs as primers.
As a result of comparison with the cDNA sequence of DAM10 (SEQ ID NO: 2), it was revealed that the obtained DNA fragment contained the 5 'upstream region of the first exon. Also, as a result of identifying the transcription start site of ADAM10 mRNA by 5 ′ RACE analysis, translation initiation codon A was found.
TG revealed that it was 239 bases upstream. No TATA box was found near the transcription start site. The GC box was located between -500 and -509 from the translation start point. Five AP2 and two Sp1 binding site-like sequences were found in the 5 'upstream region of ADAM10.
【００１１】［実施例2］PCRによる5'上流領域の増幅、
サブクローニングおよびシーケンシング 実施例１で得たフラグメントの配列を基に、配列番号６
と配列番号７をプライマーとし、上記ヒトゲノムDNAを
テンプレートとしてPCRを行った。得られたDNA断片をい
ったんpT7Blue(R)Tベクターへligationした。作製した
プラスミドはpT7Blue-ADMA981とし、塩基配列を確認し
た。[Example 2] Amplification of 5 'upstream region by PCR,
Subcloning and sequencing Based on the sequence of the fragment obtained in Example 1, SEQ ID NO: 6
And SEQ ID NO: 7 as primers, and PCR was performed using the above human genomic DNA as a template. The obtained DNA fragment was once ligated to the pT7Blue (R) T vector. The prepared plasmid was designated as pT7Blue-ADMA981, and the nucleotide sequence was confirmed.
【００１２】［実施例３］PLAPレポーター遺伝子プラス
ミドの構築 PLAPベクター (Mol. Pharmacol., 49, 860(1996))のKpn
I/Hind IIIにKpn I,Mlu I, Nhe I, Srf I, Xho I及び
Hind IIIのサイトを含むリンカーを挿入したベクターを
作製した(p-Basic-PLAPベクター）。実施例２で得たプ
ラスミドpT7Blue-ADAM981をKpn IとXho Iで消化し、pBa
sic-PLAPベクターのKpn I/Xho Iサイトに挿入して、ヒ
トADAM10のレポーターベクターp-981-PLAPを構築した。
また、実施例２で得たベクターpT7Blue-ADAM981のプロ
モーター領域の一部を欠失したベクターp-523-PLAP、p-
436-PLAPおよびp-284-PLAPはそれぞれ以下の様に作製し
た。pT7Blue-ADAM981をNdeＩ/Eco 47 III、NdeＩ/Stu I
あるいはNdeＩ/Nsp Vで消化し、平滑末端化して連結し
た。作製された３種のプラスミドをそれぞれpT7Blue(R)
Tベクター上のXba I及びヒトADAM10の5' 領域に導入し
たXho Iサイトで消化し、p-Basic-PLAPベクターのNhe I
/Xhoサイトに連結し、p-523-PLAP、p-436-PLAPおよびp-
284-PLAPを作製した（図１に示す）。Example 3 Construction of PLAP Reporter Gene Plasmid Kpn of PLAP Vector (Mol. Pharmacol., 49 , 860 (1996))
I / Hind III has Kpn I, Mlu I, Nhe I, Srf I, Xho I and
A vector into which a linker containing a Hind III site was inserted was prepared (p-Basic-PLAP vector). The plasmid pT7Blue-ADAM981 obtained in Example 2 was digested with KpnI and XhoI, and
The sic-PLAP vector was inserted into the KpnI / XhoI site to construct a human ADAM10 reporter vector p-981-PLAP.
In addition, vectors p-523-PLAP, p-523, in which a part of the promoter region of the vector pT7Blue-ADAM981 obtained in Example 2 was deleted.
436-PLAP and p-284-PLAP were prepared as follows, respectively. pT7Blue-ADAM981 was converted to NdeI / Eco 47 III, NdeI / Stu I
Alternatively, it was digested with NdeI / NspV, blunt-ended, and ligated. Each of the three plasmids thus prepared was used as pT7Blue (R)
Digested with XbaI on T vector and XhoI site introduced into 5 'region of human ADAM10, and NheI of p-Basic-PLAP vector
/ Xho site, p-523-PLAP, p-436-PLAP and p-
284-PLAP was produced (shown in FIG. 1).
【００１３】［実施例４］形質転換、培養およびPLAP活
性の測定 ブタ軟骨細胞をブタ関節より調製し、ダルベッコ・モデ
ィファイ・イーグル(Dulbecco modify Eagle)液とハムF
12液を1:1に混合した液に10％ウシ胎児血清を添加して
培養した。また、NIH3T3細胞は10％ウシ胎児血清を添加
したダルベッコ・モディファイ・イーグル(Dulbecco mo
dify Eagle)液で培養した。PLAPのアッセイは後藤(Got
o, M.)らの方法により行った(Mol. Pharmacol., 49, 86
0(1996))。細胞を、６ウェルプレートに1-3×105個の濃
度で蒔き、24時間培養した。プロモーター領域の一部を
欠失したベクターの転写活性を解析するために、培養し
た細胞に、２μgのp-981-PLAP及び一部を欠失したp-523
-PLAP、p-436-PLAP、p-284-PLAPと、0.5μgのコントロ
ールプラスミド（SV40プロモーター下流にβガラクトシ
ダーゼのコード領域を含むプラスミド：形質転換効率を
計測）を形質転換試薬FuGENE6（ロシュ・ダイアグノス
ティクス株式会社）と混和して添加して48時間培養し
た。血清に含まれるアルカリンフォスファターゼを失活
させるため、65℃で20分間熱処理した培養上清10μl
を、50μl の0.28 M Na2CO3-NaHCO3緩衝液（pH 10.0,8.
0 mM MgSO4含有）に添加し、最後に50μl アルカリンフ
ォスファターゼ基質（CDP-star、Tropix社）を添加し
た。１時間の反応後、PLAP活性をマイクロプレートリー
ダー（Wallac Eg & G社）で化学発光を検出してアルカ
リフォスファターゼ活性を測定した。図２に、ポジティ
ブコントロールであるpSV-PLAPのPLAP活性を100%とした
時の各プロモーターのPLAP活性を示す。NIH3T3細胞にお
いてはp-981-PLAP、p-523-PLAPおよびp-436-PLAPは強い
PLAP活性を示し、p-284-PLAPは弱いPLAP活性を示した
が、ネガティブコントロールであるp-Basic-PLAPとは識
別できる程度の活性を示した。一方、ブタ軟骨細胞にお
いてはp-981-PLAPは最も強いPLAP活性を示し、プロモー
ターの長さを短くするにつれてPLAP活性は低下し、p-28
4-PLAPはpBasic-PLAPと識別できるほどの活性も示さな
かった。また、NIH3T3細胞ではp-981-PLAPのPLAP活性は
ポジティブコントロールであるpSV-PLAPの29%程度なの
に対し、ブタ軟骨細胞では330%と強い活性を示した。こ
れらの結果は、我々がクローニングしたヒトADAM10の5'
領域がプロモーター活性を有すること、このプロモータ
ーがブタ軟骨細胞で強いプロモーター活性を有し、組織
特異性が認められることを示している。Example 4 Transformation, Culture and Measurement of PLAP Activity Porcine chondrocytes were prepared from porcine joints, and Dulbecco modify Eagle solution and Ham F
10% fetal bovine serum was added to a liquid obtained by mixing 12 liquids at a ratio of 1: 1 and cultured. In addition, NIH3T3 cells were prepared from Dulbecco's modified Eagle supplemented with 10% fetal bovine serum.
dify Eagle) solution. Goto (Got)
o, M.) et al. (Mol. Pharmacol., 49 , 86).
0 (1996)). Cells were seeded in 6-well plates at a concentration of 1-3 × 10 5 and cultured for 24 hours. In order to analyze the transcriptional activity of the vector lacking a part of the promoter region, 2 μg of p-981-PLAP and p-523 partially lacking were added to the cultured cells.
-PLAP, p-436-PLAP, p-284-PLAP and 0.5 μg of a control plasmid (a plasmid containing the β-galactosidase coding region downstream of the SV40 promoter: measuring the transformation efficiency) were transformed with the transformation reagent FuGENE6 (Roche Diagnostics). Nostics Co., Ltd.) and cultured for 48 hours. 10 μl of culture supernatant heat-treated at 65 ° C for 20 minutes to inactivate alkaline phosphatase contained in serum
With 50 μl of 0.28 M Na 2 CO 3 -NaHCO 3 buffer (pH 10.0,8.
0 mM MgSO 4 ) and finally 50 μl alkaline phosphatase substrate (CDP-star, Tropix). After the reaction for 1 hour, alkaline phosphatase activity was measured by detecting chemiluminescence of PLAP activity using a microplate reader (Wallac Eg & G). FIG. 2 shows the PLAP activity of each promoter when the PLAP activity of pSV-PLAP, which is a positive control, is set to 100%. P-981-PLAP, p-523-PLAP and p-436-PLAP are strong in NIH3T3 cells
It showed PLAP activity, and p-284-PLAP showed weak PLAP activity, but showed an activity that could be distinguished from p-Basic-PLAP as a negative control. On the other hand, in porcine chondrocytes, p-981-PLAP showed the strongest PLAP activity, and the PLAP activity decreased as the length of the promoter was shortened.
4-PLAP did not show any activity that could be distinguished from pBasic-PLAP. In NIH3T3 cells, the PLAP activity of p-981-PLAP was about 29% of the positive control pSV-PLAP, whereas the porcine chondrocytes showed a strong activity of 330%. These results indicate that we cloned the 5 'human ADAM10
This indicates that the region has promoter activity, and that this promoter has strong promoter activity in porcine chondrocytes and has tissue specificity.
【００１４】[0014]
【配列表】 <110> Eisai Co., Ltd. <120> ADAM10 promoter <130> EP99TH1101 <160> 7 <170> PatentIn Ver. 2.0 <210> 1 <211> 981 <212> DNA <213> Human <400> 1 aagctttaag aagttgggga gaagatgtgc gtggattcgc ttcaaagatg tgacagagag 60 aaaagaggac attttctgga ctggagattc aagacagtct ggttcaggtt gcatgctttt 120 gcaaaggcta aagccttagt ctgtgacata aatttgtaaa gagctcggaa aattacatct 180 cggacatggt gtcttttttt cctgctgctt gaaggtcttt gcatttatgt tcgcatcact 240 ggggcagctg ccgctataat tactgcttgt tgggactttt tgtttacttg gtagctgtgg 300 tgcaccaaga gaggcagaaa aagaagaaaa aaaacctctg ttacttgtga ctttaagaag 360 tcgaaagcag ccctgcttac atcttccacg gaccatttta gcccaaggga aggtcctcag 420 cagctctaac acgtagcgga gcactatctc cgcgtaggag cgctcccgcc ccggggcggg 480 accaggacaa accccgcctc ccaagcccaa tcccagctct ccgccggcgg acaggaagcg 540 gcggcaggcc tagcagcacg ggaaccgtcc cccgcgcgca tgcgcgcgcc cctgaagcgc 600 ctgggggacg ggtaggggcg ggaggtaggg gcgcggctcc gcgtgccagt tgggtgcccg 660 cgcgtcacgt ggtgaggaag gaagcggagg tctgagtttc gagggagggg gggagagaag 720 agggaacgag caagggaagg aaagcgggga aaggaggaag gaaacgaacg agggggaggg 780 aggtccctgt tttggaggag ctaggagcgt tgccggcccc tgaagtggag cgagagggag 840 gtgcttcgcc gtttctcctg ccaggggagg tcccggcttc ccgtggaggc tccggaccaa 900 gccccttcag cttctccctc cggatcgatg tgctgctgtt aacccgtgag gaggcggcgg 960 cggcggcagc ggcagcggaa g 981[Sequence List] <110> Eisai Co., Ltd. <120> ADAM10 promoter <130> EP99TH1101 <160> 7 <170> PatentIn Ver. 2.0 <210> 1 <211> 981 <212> DNA <213> Human < 400> 1 aagctttaag aagttgggga gaagatgtgc gtggattcgc ttcaaagatg tgacagagag 60 aaaagaggac attttctgga ctggagattc aagacagtct ggttcaggtt gcatgctttt 120 gcaaaggcta aagccttagt ctgtgacata aatttgtaaa gagctcggaa aattacatct 180 cggacatggt gtcttttttt cctgctgctt gaaggtcttt gcatttatgt tcgcatcact 240 ggggcagctg ccgctataat tactgcttgt tgggactttt tgtttacttg gtagctgtgg 300 tgcaccaaga gaggcagaaa aagaagaaaa aaaacctctg ttacttgtga ctttaagaag 360 tcgaaagcag ccctgcttac atcttccacg gaccatttta gcccaaggga aggtcctcag 420 cagctctaac acgtagcgga gcactatctc cgcgtaggag cgctcccgcc ccggggcggg 480 accaggacaa accccgcctc ccaagcccaa tcccagctct ccgccggcgg acaggaagcg 540 gcggcaggcc tagcagcacg ggaaccgtcc cccgcgcgca tgcgcgcgcc cctgaagcgc 600 ctgggggacg ggtaggggcg ggaggtaggg gcgcggctcc gcgtgccagt tgggtgcccg 660 cgcgtcacgt ggtgaggaag gaagcggagg tctgagtttc gagggagggg gggagagaag 720 aggg aacgag caagggaagg aaagcgggga aaggaggaag gaaacgaacg agggggaggg 780 aggtccctgt tttggaggag ctaggagcgt tgccggcccc tgaagtggag cgagagggag 840 gtgcttcgcc gtttctcctg ccaggggagg tcccggcttc ccgtggaggc tccggaccaa 900 gccccttcag cttctccctc cggatcgatg tgctgctgtt aacccgtgag gaggcggcgg 960 cggcggcagc ggcagcggaa g 981
【００１５】 <210> 2 <211> 3410 <212> DNA <213> Human <220> <221> CDS <222> (470)..(2716) <400> 2 gaattcgagg atccgggtac catgggcggc ggcaggccta gcagcacggg aaccgtcccc 60 cgcgcgcatg cgcgcgcccc tgaagcgcct gggggacggg tatgggcggg aggtaggggc 120 gcggctccgc gtgccagttg ggtgcccgcg cgtcacgtgg tgaggaagga ggcggaggtc 180 tgagtttcga gggagggggg gagagaagag ggaacgagca agggaaggaa agcggggaaa 240 ggaggaagga aacgaacgag ggggagggag gtccctgttt tggaggagct aggagcgttg 300 ccggcccctg aagtggagcg agagggaggt gcttcgccgt ttctcctgcc aggggaggtc 360 ccggcttccc gtggaggctc cggaccaagc cccttcagct tctccctccg gatcgatgtg 420 ctgctgttaa cccgtgagga ggcggcggcg gcggcagcgg cagcggaag atg gtg ttg 478 Met Val Leu 1 ctg aga gtg tta att ctg ctc ctc tcc tgg gcg gcg ggg atg gga ggt 526 Leu Arg Val Leu Ile Leu Leu Leu Ser Trp Ala Ala Gly Met Gly Gly 5 10 15 cag tat ggg aat cct tta aat aaa tat atc aga cat tat gaa gga tta 574 Gln Tyr Gly Asn Pro Leu Asn Lys Tyr Ile Arg His Tyr Glu Gly Leu 20 25 30 35 tct tac aat gtg gat tca tta cac caa aaa cac cag cgt gcc aaa aga 622 Ser Tyr Asn Val Asp Ser Leu His Gln Lys His Gln Arg Ala Lys Arg 40 45 50 gca gtc tca cat gaa gac caa ttt tta cgt cta gat ttc cat gcc cat 670 Ala Val Ser His Glu Asp Gln Phe Leu Arg Leu Asp Phe His Ala His 55 60 65 gga aga cat ttc aac cta cga atg aag agg gac act tcc ctt ttc agt 718 Gly Arg His Phe Asn Leu Arg Met Lys Arg Asp Thr Ser Leu Phe Ser 70 75 80 gat gaa ttt aaa gta gaa aca tca aat aaa gta ctt gat tat gat acc 766 Asp Glu Phe Lys Val Glu Thr Ser Asn Lys Val Leu Asp Tyr Asp Thr 85 90 95 tct cat att tac act gga cat att tat ggt gaa gaa gga agt ttt agc 814 Ser His Ile Tyr Thr Gly His Ile Tyr Gly Glu Glu Gly Ser Phe Ser 100 105 110 115 cat ggg tct gtt att gat gga aga ttt gaa gga ttc atc cag act cgt 862 His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile Gln Thr Arg 120 125 130 ggt ggc aca ttt tat gtt gag cca gca gag aga tat att aaa gac cga 910 Gly Gly Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile Lys Asp Arg 135 140 145 act ctg cca ttt cac tct gtc att tat cat gaa gat gat att aac tat 958 Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp Ile Asn Tyr 150 155 160 ccc cat aaa tac ggt cct cag ggg ggc tgt gca gat cat tca gta ttt 1006 Pro His Lys Tyr Gly Pro Gln Gly Gly Cys Ala Asp His Ser Val Phe 165 170 175 gaa aga atg agg aaa tac cag atg act ggt gta gag gaa gta aca cag 1054 Glu Arg Met Arg Lys Tyr Gln Met Thr Gly Val Glu Glu Val Thr Gln 180 185 190 195 ata cct caa gaa gaa cat gct gct aat ggt cca gaa ctt ctg agg aaa 1102 Ile Pro Gln Glu Glu His Ala Ala Asn Gly Pro Glu Leu Leu Arg Lys 200 205 210 aaa cgt aca act tca gct gaa aaa aat act tgt cag ctt tat att cag 1150 Lys Arg Thr Thr Ser Ala Glu Lys Asn Thr Cys Gln Leu Tyr Ile Gln 215 220 225 act gat cat ttg ttc ttt aaa tat tac gga aca cga gaa gct gtg att 1198 Thr Asp His Leu Phe Phe Lys Tyr Tyr Gly Thr Arg Glu Ala Val Ile 230 235 240 gcc cag ata tcc agt cat gtt aaa gcg att gat aca att tac cag acc 1246 Ala Gln Ile Ser Ser His Val Lys Ala Ile Asp Thr Ile Tyr Gln Thr 245 250 255 aca gac ttc tcc gga atc cgt aac atc agt ttc atg gtg aaa cgc ata 1294 Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val Lys Arg Ile 260 265 270 275 aga atc aat aca act gct gat gag aag gac cct aca aat cct ttc cgt 1342 Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn Pro Phe Arg 280 285 290 ttc cca aat att ggt gtg gag aag ttt ctg gaa ttg aat tct gag cag 1390 Phe Pro Asn Ile Gly Val Glu Lys Phe Leu Glu Leu Asn Ser Glu Gln 295 300 305 aat cat gat gac tac tgt ttg gcc tat gtc ttc aca gac cga gat ttt 1438 Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp Arg Asp Phe 310 315 320 gat gat ggc gta ctt ggt ctg gct tgg gtt gga gca cct tca gga agc 1486 Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro Ser Gly Ser 325 330 335 tct gga gga ata tgt gaa aaa agt aaa ctc tat tca gat ggt aag aag 1534 Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp Gly Lys Lys 340 345 350 355 aag tcc tta aac act gga att att act gtt cag aac tat ggg tct cat 1582 Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr Gly Ser His 360 365 370 gta cct ccc aaa gtc tct cac att act ttt gct cac gaa gtt gga cat 1630 Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu Val Gly His 375 380 385 aac ttt gga tcc cca cat gat tct gga aca gag tgc aca cca gga gaa 1678 Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr Pro Gly Glu 390 395 400 tct aag aat ttg ggt caa aaa gaa aat ggc aat tac atc atg tat gca 1726 Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile Met Tyr Ala 405 410 415 aga gca aca tct ggg gac aaa ctt aac aac aat aaa ttc tca ctc tgt 1774 Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe Ser Leu Cys 420 425 430 435 agt att aga aat ata agc caa gtt ctt gag aag aag aga aac aac tgt 1822 Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg Asn Asn Cys 440 445 450 ttt gtt gaa tct ggc caa cct att tgt gga aat gga atg gta gaa caa 1870 Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met Val Glu Gln 455 460 465 ggt gaa gaa tgt gat tgt ggc tat agt gac cag tgt aaa gat gaa tgc 1918 Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys Asp Glu Cys 470 475 480 tgc ttc gat gca aat caa cca gag gga aga aaa tgc aaa ctg aaa cct 1966 Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys Leu Lys Pro 485 490 495 ggg aaa cag tgc agt cca agt caa ggt cct tgt tgt aca gca cag tgt 2014 Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr Ala Gln Cys 500 505 510 515 gca ttc aag tca aag tct gag aag tgt cgg gat gat tca gac tgt gca 2062 Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser Asp Cys Ala 520 525 530 agg gaa gga ata tgt aat ggc ttc aca gct ctc tgc cca gca tct gac 2110 Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro Ala Ser Asp 535 540 545 cct aaa cca aac ttc aca gac tgt aat agg cat aca caa gtg tgc att 2158 Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln Val Cys Ile 550 555 560 aat ggg caa tgt gca ggt tct atc tgt gag aaa tat ggc tta gag gag 2206 Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly Leu Glu Glu 565 570 575 tgt acg tgt gcc agt tct gat ggc aaa gat gat aaa gaa tta tgc cat 2254 Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu Leu Cys His 580 585 590 595 gta tgc tgt atg aag aaa atg gac cca tca act tgt gcc agt aca ggg 2302 Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala Ser Thr Gly 600 605 610 tct gtg cag tgg agt agg cac ttc agt ggt cga acc atc acc ctg caa 2350 Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile Thr Leu Gln 615 620 625 cct gga tcc cct tgc aac gat ttt aga ggt tac tgt gat gtt ttc atg 2398 Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp Val Phe Met 630 635 640 cgg tgc aga tta gta gat gct gat ggt cct cta gct agg ctt aaa aaa 2446 Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg Leu Lys Lys 645 650 655 gca att ttt agt cca gag ctc tat gaa aac att gct gaa tgg att gtg 2494 Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu Trp Ile Val 660 665 670 675 gct cat tgg tgg gca gta tta ctt atg gga att gct ctg atc atg cta 2542 Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu Ile Met Leu 680 685 690 atg gct gga ttt att aag ata tgc agt gtt cat act cca agt agt aat 2590 Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro Ser Ser Asn 695 700 705 cca aag ttg cct cct cct aaa cca ctt cca ggc act tta aag agg agg 2638 Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu Lys Arg Arg 710 715 720 aga cct cca cag ccc att cag caa ccc cag cgt cag cgg ccc cga gag 2686 Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg Pro Arg Glu 725 730 735 agt tat caa atg gga cac atg aga cgc taa ctgcagcttt tgccttggtt 2736 Ser Tyr Gln Met Gly His Met Arg Arg 740 745 cttcctagtg cctacaatgg gaaaacttca ctccaaagag aaacctatta agtcatcatc 2796 tccaaactaa accctcacaa gtaacagttg aagaaaaaat ggcaagagat catatcctca 2856 gaccaggtgg aattacttaa attttaaagc ctgaaaattc caatttgggg gtgggaggtg 2916 gaaaaggaac ccaattttct tatgaacaga tatttttaac ttaatggcac aaagtcttag 2976 aatattatta tgtgccccgt gttccctgtt cttcgttgct gcattttctt cacttgcagg 3036 caaacttggc tctcaataaa cttttaccac aaattgaaat aaatatattt ttttcaactg 3096 ccaatcaagg ctaggaggct cgaccacctc aacattggag acatcacttg ccaatgtaca 3156 taccttgtta tatgcagaca tgtatttctt acgtacactg tacttctgtg tgcaattgta 3216 aacagaaatt gcaatatgga tgtttctttg tattataaaa tttttccgct cttaattaaa 3276 aattactgtt taattgacat actcaggata acagagaatg gtggtattca gtggtccagg 3336 attctgtaat gctttacaca ggcagttttg aaatgaaaat caatttaccc catggtaccc 3396 ggatcctcga attc 3410<210> 2 <211> 3410 <212> DNA <213> Human <220> <221> CDS <222> (470) .. (2716) <400> 2 gaattcgagg atccgggtac catgggcggc ggcaggccta gcagcacggg aaccgtcccc 60 cgcgcgcatg cgcgcgcccc tgaagcgcct gggggacggg tatgggcggg aggtaggggc 120 gcggctccgc gtgccagttg ggtgcccgcg cgtcacgtgg tgaggaagga ggcggaggtc 180 tgagtttcga gggagggggg gagagaagag ggaacgagca agggaaggaa agcggggaaa 240 ggaggaagga aacgaacgag ggggagggag gtccctgttt tggaggagct aggagcgttg 300 ccggcccctg aagtggagcg agagggaggt gcttcgccgt ttctcctgcc aggggaggtc 360 ccggcttccc gtggaggctc cggaccaagc cccttcagct tctccctccg gatcgatgtg 420 ctgctgttaa cccgtgagga ggcggcggcg gcggcagcgg cagcggaag atg gtg ttg 478 Met Val Leu 1 ctg aga gtg tta att ctg ctc ctc tcc tgg gcg gcg ggg atg gga ggt 526 Leu Arg Val Leu Ile Leu Leu Leu Ser Trp Ala Ala Gly Met Gly Gly 5 10 15 cag tat ggg aat cct tta aat aaa tat at cat tat gaa gga tta 574 Gln Tyr Gly Asn Pro Leu Asn Lys Tyr Ile Arg His Tyr Glu Gly Leu 20 25 30 35 tct tac aat gtg gat tca tta cac caa aaa cac cag cgt gcc aaa aga 622 Ser Tyr Asn Val Asp Ser Leu His Gln Lys His Gln Arg Ala Lys Arg 40 45 50 gca gtc tca cat gaa gac caa ttt tta cgt cta gat ttc cat gcc cat 670 Ala Val Ser His Glu Asp Gln Phe Leu Arg Leu Asp Phe His Ala His 55 60 65 gga aga cat ttc aac cta cga atg aag agg gac act tcc ctt ttc agt 718 Gly Arg His Phe Asn Leu Arg Met Lys Arg Asp Thr Ser Leu Phe Ser 70 75 80 gat gaa ttt aaa gta gaa aca tca aat aaa gta ctt gat tat gat acc 766 Asp Glu Phe Lys Val Glu Thr Ser Asn Lys Val Leu Asp Tyr Asp Thr 85 90 95 tct cat att tac act gga cat att tat ggt gaa gaa gga agt ttt agc 814 Ser His Ile Tyr Thr Gly His Ile Tyr Gly Glu Glu Gly Ser Phe Ser 100 105 110 115 cat ggg tct gtt att gat gga aga ttt gaa gga ttc atc cag act cgt 862 His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile Gln Thr Arg 120 125 130 ggt ggc aca ttt tat gtt gag cca gca gag aga tat att aaa gac cga 910 Gly Gly Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile Lys Asp Arg 135 140 145 act ctg cca ttt cac tct gtc att tat cat gaa gat gat att aac tat 958 Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp Ile Asn Tyr 150 155 160 ccc cat aaa tac ggt cct cag ggg ggc tgt gca gat cat tca gta ttt 1006 Pro His Lys Tyr Gly Pro Gln Gly Gly Cys Ala Asp His Ser Val Phe 165 170 175 gaa aga atg agg aaa tac cag atg act ggt gta gag gaa gta aca cag 1054 Glu Arg Met Arg Lys Tyr Gln Met Thr Gly Val Glu Glu Val Thr Gln 180 185 190 195 ata cct caa gaa gaa cat gct gct aat ggt cca gaa ctt ctg agg aaa 1102 Ile Pro Gln Glu Glu His Ala Ala Asn Gly Pro Glu Leu Leu Arg Lys 200 205 210 aaa cgt aca act tca gct gaa aaa aat act tgt cag ctt tat att cag 1150 Lys Arg Thr Thr Ser Ala Glu Lys Asn Thr Cys Gln Leu Tyr Ile Gln 215 220 225 act gat cat ttg ttc ttt aaa tat tac gga aca cga gaa gct gtg att 1198 Thr Asp His Leu Phe Phe Lys Tyr Tyr Gly Thr Arg Glu Ala Val Ile 230 235 240 gcc cag ata tcc agt cat gtt aaa gcg att gat aca att tac cag acc 1246 Ala Gln Ile Ser Ser His Val Lys Ala Ile Asp Thr Ile Tyr Gln Thr 245 250 255 aca gac ttc tcc gga a tc cgt aac atc agt ttc atg gtg aaa cgc ata 1294 Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val Lys Arg Ile 260 265 270 275 aga atc aat aca act gct gat gag aag gac cct aca aat cct ttc cgt 134g Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn Pro Phe Arg 280 285 290 ttc cca aat att ggt gtg gag aag ttt ctg gaa ttg aat tct gag cag 1390 Phe Pro Asn Ile Gly Val Glu Lys Phe Leu Glu Leu Asn Ser Glu Gln 295 300 305 aat cat gat gac tac tgt ttg gcc tat gtc ttc aca gac cga gat ttt 1438 Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp Arg Asp Phe 310 315 320 gat gat ggc gta ctt ggt ctg gct tgg gga gca cct tca gga agc 1486 Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro Ser Gly Ser 325 330 335 tct gga gga ata tgt gaa aaa agt aaa ctc tat tca gat ggt aag aag 1534 Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp Gly Lys Lys 340 345 350 355 aag tcc tta aac act gga att att act gtt cag aac tat ggg tct cat 1582 Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr Gly Ser His 360 365 37 0 gta cct ccc aaa gtc tct cac att act ttt gct cac gaa gtt gga cat 1630 Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu Val Gly His 375 380 385 aac ttt gga tcc cca cat gat tct gga aca gag tgc aca cca gga gaa 1678 Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr Pro Gly Glu 390 395 400 tct aag aat ttg ggt caa aaa gaa aat ggc aat tac atc atg tat gca 1726 Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile Met Tyr Ala 405 410 415 aga gca aca tct ggg gac aaa ctt aac aac aat aaa ttc tca ctc tgt 1774 Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe Ser Leu Cys 420 425 430 435 435 agt att aga aat ata agc caa gtt ctt gag aag aag taga 1822 Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg Asn Asn Cys 440 445 450 ttt gtt gaa tct ggc caa cct att tgt gga aat gga atg gta gaa caa 1870 Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met Val Glu Gln 455 460 465 ggt gaa gaa tgt gat tgt ggc tat agt gac cag tgt aaa gat gaa tgc 1918 Gly Glu Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys As Glu Cys 470 475 480 tgc ttc gat gca aat caa cca gag gga aga aaa tgc aaa ctg aaa cct 1966 Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys Leu Lys Pro 485 490 495 ggg aaa cag tgc agtc agtca cct tgt tgt aca gca cag tgt 2014 Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr Ala Gln Cys 500 505 510 515 gca ttc aag tca aag tct gag aag tgt cgg gat gat tca gac tgt gca 2062 Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser Asp Cys Ala 520 525 530 agg gaa gga ata tgt aat ggc ttc aca gct ctc tgc cca gca tct gac 2110 Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro Ala Ser As545 535 cct aaa cca aac ttc aca gac tgt aat agg cat aca caa gtg tgc att 2158 Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln Val Cys Ile 550 555 560 aat ggg caa tgt gca ggt tct atc tgt gag aaa tat ggctta gag gag 2206 Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly Leu Glu Glu 565 570 575 tgt acg tgt gcc agt tct gat ggc aaa gat gat aaa gaa tta tgc cat 2254 Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu Leu Cys His 580 585 590 595 gta tgc tgt atg aag aaa atg gac cca tca act tgt gcc agt aca ggg 2302 Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala Ser Thr Gly 600 605 610 tct gtg cag tgg agt agg cac ttc agt ggt cga acc atc acc ctg caa 2350 Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile Thr Leu Gln 615 620 625 cct gga tcc cct tgc aac gat ttt aga ggt tac tgt gat gtt ttc atg 2398 Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp Val Phe Met 630 635 640 cgg tgc aga gta gat gct gat ggt cct cta gct agg ctt aaa aaa 2446 Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg Leu Lys Lys 645 650 655 gca att ttt agt cca gag ctc tat gaa aac att gct gaa tgg att gtg 2494 Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu Trp Ile Val 660 665 670 675 gct cat tgg cgg gca gta tta atg gga att gct ctg atc atg cta 2542 Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu Ile Met Leu 680 685 690 atg gct gga ttt att aag ata tgc agt gtt cat act cca agt agt aat 2590 Met Ala Gly P he Ile Lys Ile Cys Ser Val His Thr Pro Ser Ser Asn 695 700 705 cca aag ttg cct cct cct aaa cca ctt cca ggc act tta aag agg agg 2638 Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu Lys Arg Arg 710 715 720 aga cct cca cag ccc att cag caa ccc cag cgt cag cgg ccc cga gag 2686 Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg Pro Arg Glu 725 730 735 agt tat caa atg gga cac atg aga cgc taa tggccgtt 2736 Ser Tyr Gln Met Gly His Met Arg Arg 740 745 cttcctagtg cctacaatgg gaaaacttca ctccaaagag aaacctatta agtcatcatc 2796 tccaaactaa accctcacaa gtaacagttg aagaaaaaat ggcaagagat catatcctca 2856 gaccaggtgg aattacttaa attttaaagc ctgaaaattc caatttgggg gtgggaggtg 2916 gaaaaggaac ccaattttct tatgaacaga tatttttaac ttaatggcac aaagtcttag 2976 aatattatta tgtgccccgt gttccctgtt cttcgttgct gcattttctt cacttgcagg 3036 caaacttggc tctcaataaa cttttaccac aaattgaaat aaatatattt ttttcaactg 3096 ccaatcaagg ctaggaggct cgaccacctc aacattggag acatcacttg ccaatgtaca 3156 taccttgtta tatgcagaca tgtatttctt acgtac actg tacttctgtg tgcaattgta 3216 aacagaaatt gcaatatgga tgtttctttg tattataaaa tttttccgct cttaattaaa 3276 aattactgtt taattgacat actcaggata acagagaatg gtggtattca gtggtccagg 3336 attctgtaat gctttacagatccatccatccatccatcac gatcattagcac gatcattagcac gatcattagcac gatcattagcac gatcattagcac gagtacctg
【００１６】 <210> 3 <211> 748 <212> PRT <213> Human <400> 3 Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu Ser Trp Ala Ala Gly 1 5 10 15 Met Gly Gly Gln Tyr Gly Asn Pro Leu Asn Lys Tyr Ile Arg His Tyr 20 25 30 Glu Gly Leu Ser Tyr Asn Val Asp Ser Leu His Gln Lys His Gln Arg 35 40 45 Ala Lys Arg Ala Val Ser His Glu Asp Gln Phe Leu Arg Leu Asp Phe 50 55 60 His Ala His Gly Arg His Phe Asn Leu Arg Met Lys Arg Asp Thr Ser 65 70 75 80 Leu Phe Ser Asp Glu Phe Lys Val Glu Thr Ser Asn Lys Val Leu Asp 85 90 95 Tyr Asp Thr Ser His Ile Tyr Thr Gly His Ile Tyr Gly Glu Glu Gly 100 105 110 Ser Phe Ser His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile 115 120 125 Gln Thr Arg Gly Gly Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile 130 135 140 Lys Asp Arg Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp 145 150 155 160 Ile Asn Tyr Pro His Lys Tyr Gly Pro Gln Gly Gly Cys Ala Asp His 165 170 175 Ser Val Phe Glu Arg Met Arg Lys Tyr Gln Met Thr Gly Val Glu Glu 180 185 190 Val Thr Gln Ile Pro Gln Glu Glu His Ala Ala Asn Gly Pro Glu Leu 195 200 205 Leu Arg Lys Lys Arg Thr Thr Ser Ala Glu Lys Asn Thr Cys Gln Leu 210 215 220 Tyr Ile Gln Thr Asp His Leu Phe Phe Lys Tyr Tyr Gly Thr Arg Glu 225 230 235 240 Ala Val Ile Ala Gln Ile Ser Ser His Val Lys Ala Ile Asp Thr Ile 245 250 255 Tyr Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val 260 265 270 Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn 275 280 285 Pro Phe Arg Phe Pro Asn Ile Gly Val Glu Lys Phe Leu Glu Leu Asn 290 295 300 Ser Glu Gln Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp 305 310 315 320 Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro 325 330 335 Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp 340 345 350 Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr 355 360 365 Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu 370 375 380 Val Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr 385 390 395 400 Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile 405 410 415 Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe 420 425 430 Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg 435 440 445 Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met 450 455 460 Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys 465 470 475 480 Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys 485 490 495 Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr 500 505 510 Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser 515 520 525 Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro 530 535 540 Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln 545 550 555 560 Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly 565 570 575 Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu 580 585 590 Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala 595 600 605 Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile 610 615 620 Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp 625 630 635 640 Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg 645 650 655 Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu 660 665 670 Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu 675 680 685 Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro 690 695 700 Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu 705 710 715 720 Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg 725 730 735 Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg 740 745<210> 3 <211> 748 <212> PRT <213> Human <400> 3 Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu Ser Trp Ala Ala Gly 1 5 10 15 Met Gly Gly Gln Tyr Gly Asn Pro Leu Asn Lys Tyr Ile Arg His Tyr 20 25 30 Glu Gly Leu Ser Tyr Asn Val Asp Ser Leu His Gln Lys His Gln Arg 35 40 45 Ala Lys Arg Ala Val Ser His Glu Asp Gln Phe Leu Arg Leu Asp Phe 50 55 60 His Ala His Gly Arg His Phe Asn Leu Arg Met Lys Arg Asp Thr Ser 65 70 75 80 Leu Phe Ser Asp Glu Phe Lys Val Glu Thr Ser Asn Lys Val Leu Asp 85 90 95 Tyr Asp Thr Ser His Ile Tyr Thr Gly His Ile Tyr Gly Glu Glu Gly 100 105 110 Ser Phe Ser His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile 115 120 125 Gln Thr Arg Gly Gly Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile 130 135 140 Lys Asp Arg Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp 145 150 155 160 Ile Asn Tyr Pro His Lys Tyr Gly Pro Gln Gly Gly Cys Ala Asp His 165 170 175 Ser Val Phe Glu Arg Met Arg Lys Tyr Gln Met Thr Gly Val Glu Glu 180 185 190 Val Thr Gln Ile Pro Gln Glu Glu His Ala Ala Asn Gly Pro Glu Leu 195 200 205 Leu Arg Lys Lys Arg Thr Thr Ser Ala Glu Lys Asn Thr Cys Gln Leu 210 215 220 Tyr Ile Gln Thr Asp His Leu Phe Phe Lys Tyr Tyr Gly Thr Arg Glu 225 230 235 240 Ala Val Ile Ala Gln Ile Ser Ser His Val Lys Ala Ile Asp Thr Ile 245 250 255 Tyr Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val 260 265 270 Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn 275 280 285 Pro Phe Arg Phe Pro Asn Ile Gly Val Glu Lys Phe Leu Glu Leu Asn 290 295 300 Ser Glu Gln Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp 305 310 315 320 Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro 325 330 335 Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp 340 345 350 Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr 355 360 365 Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu 370 375 380 Val Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr 385 390 395 400 400 Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile 405 410 415 Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe 420 425 430 Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg 435 440 445 Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met 450 455 460 Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys 465 470 475 480 Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys 485 490 495 Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr 500 505 510 Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser 515 520 525 Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro 530 535 540 Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln 545 550 555 560 Val Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly 565 570 575 Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu 580 585 590 Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala 595 600 605 Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile 610 615 620 Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp 625 630 635 640 Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg 645 650 655 Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu 660 665 670 Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu 675 680 685 Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro 690 695 700 Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu 705 710 715 720 Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg 725 730 735 Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg 740 745
【００１７】 <210> 4 <211> 26 <212> DNA <213> Human <400> 4 tcctagctcc tccaaaacag ggacct 26 <210> 5 <211> 26 <212> DNA <213> Human <400> 5 tgaagtggag cgagagggag gtgctt 26 <210> 6 <211> 30 <212> DNA <213> Human <400> 6 ggggggtacc aagctttaag aagttgggga 30 <210> 7 <211> 30 <212> DNA <213> Human <400> 7 taacactctc agcaactcga gcttccgctg 30<210> 4 <211> 26 <212> DNA <213> Human <400> 4 tcctagctcc tccaaaacag ggacct 26 <210> 5 <211> 26 <212> DNA <213> Human <400> 5 tgaagtggag cgagagggag gtgctt 26 <210> 6 <211> 30 <212> DNA <213> Human <400> 6 ggggggtacc aagctttaag aagttgggga 30 <210> 7 <211> 30 <212> DNA <213> Human <400> 7 taacactctc agcaactcga gcttccgctg 30
【００１８】[0018]
【図１】ADAM10遺伝子プロモーター領域の一部を欠失し
たベクター。FIG. 1 shows a vector in which a part of the ADAM10 gene promoter region has been deleted.
【図２】一部を欠失したADAM10遺伝子プロモーター領域
の転写活性。FIG. 2 shows the transcriptional activity of a partially deleted ADAM10 gene promoter region.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｑ 1/68 Ｃ１２Ｎ 5/00 Ａ Ｆターム(参考） 4B024 AA11 AA20 BA80 CA04 DA02 EA04 FA02 GA11 HA01 HA12 HA20 4B063 QA01 QQ42 QR33 QR59 QR62 QS25 4B065 AA90X AA90Y AA99Y AB01 BA02 CA46 CA60 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 C12N 5/00 A F term (Reference) 4B024 AA11 AA20 BA80 CA04 DA02 EA04 FA02 GA11 HA01 HA12 HA20 4B063 QA01 QQ42 QR33 QR59 QR62 QS25 4B065 AA90X AA90Y AA99Y AB01 BA02 CA46 CA60
Claims (6)
A。1. A DN comprising the nucleotide sequence of SEQ ID NO: 1.
A.
とストリンジェントな条件でハイブリダイズし、ADAM10
の転写調節を行うプロモーター活性を持ったDNA。2. A DNA comprising the nucleotide sequence of SEQ ID NO: 1.
Hybridizes with stringent conditions with ADAM10
DNA with promoter activity that regulates the transcription of DNA.
るDNAをプライマーとしてPCRにより増幅されるDNAで、A
DAM10の転写調節を行うプロモーター活性を持ったDNA。3. A DNA which is amplified by PCR using a DNA comprising a part of the nucleotide sequence of SEQ ID NO: 1 as a primer,
DNA with promoter activity that regulates the transcription of DAM10.
むベクター。A vector comprising the DNA according to any one of claims 1 to 3.
胞。5. A cell into which the vector according to claim 4 has been introduced.
とする、ADAM10遺伝子の発現調節に影響を与える化合物
をスクリーニングする方法。6. A method for screening a compound that affects the regulation of ADAM10 gene expression, comprising using the cell according to claim 5.
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| JP31214999A JP2001128677A (en) | 1999-11-02 | 1999-11-02 | Promoter regulating expression of adam10 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP31214999A JP2001128677A (en) | 1999-11-02 | 1999-11-02 | Promoter regulating expression of adam10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2001128677A true JP2001128677A (en) | 2001-05-15 |
Family
ID=18025847
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| Application Number | Title | Priority Date | Filing Date |
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| JP31214999A Pending JP2001128677A (en) | 1999-11-02 | 1999-11-02 | Promoter regulating expression of adam10 |
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| Country | Link |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899280A (en) * | 2021-04-09 | 2021-06-04 | 中国药科大学 | AD cell model established based on CRISPR/Cas9 gene editing technology and construction method and application thereof |
-
1999
- 1999-11-02 JP JP31214999A patent/JP2001128677A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899280A (en) * | 2021-04-09 | 2021-06-04 | 中国药科大学 | AD cell model established based on CRISPR/Cas9 gene editing technology and construction method and application thereof |
| CN112899280B (en) * | 2021-04-09 | 2023-11-03 | 中国药科大学 | AD cell model established based on CRISPR/Cas9 gene editing technology, and construction method and application thereof |
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