JP2904935B2 - Neurotrophic activity inhibitor - Google Patents
Neurotrophic activity inhibitorInfo
- Publication number
- JP2904935B2 JP2904935B2 JP2410164A JP41016490A JP2904935B2 JP 2904935 B2 JP2904935 B2 JP 2904935B2 JP 2410164 A JP2410164 A JP 2410164A JP 41016490 A JP41016490 A JP 41016490A JP 2904935 B2 JP2904935 B2 JP 2904935B2
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- JP
- Japan
- Prior art keywords
- cys
- tgc
- lys
- gag
- aag
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Description
【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION
【０００１】[0001]
【産業上の利用分野】本発明は、新規で有用な蛋白に関
するものであり、さらに詳しくは、ヒト脳中に存在する
神経栄養活性抑制作用を有する蛋白質に関するものであ
る。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel and useful protein, and more particularly to a protein having an inhibitory effect on neurotrophic activity existing in the human brain.
【０００２】[0002]
【従来の技術】高齢化社会の中で、老人性痴呆は、大き
な関心を集め、その予防と治療には、多くの努力が払わ
れてきた。特に、アルツハイマー（Ａｌｚｈｅｉｍｅ
ｒ）病と言われる老人性痴呆は、初老期（５０〜６０
才）に来ることが多く、その原因の究明と治療法の確立
が急がれている。 現在までに得られた知見によれば、アルツハイマー病
は、老人班、神経原線維変性などの病理学的特徴と、進
行性痴呆という臨床的特徴を有する器質性疾患であり、
ンニューロンの代謝の亢進や異常な再生が関与している
と考えられる。2. Description of the Related Art In an aging society, senile dementia has attracted great interest, and much effort has been put into its prevention and treatment. In particular, Alzheimer (Alzheimer)
r) Senile dementia, which is referred to as a disease, occurs in the elderly (50-60).
Years old), and it is urgent to investigate the cause and establish a cure. According to the findings obtained to date, Alzheimer's disease is an organic disease having pathological characteristics such as senile plaques, neurofibrillary degeneration, and clinical characteristics of progressive dementia,
It is thought that enhanced metabolism and abnormal regeneration of neurons are involved.
【０００３】しかしながら、従来、このアルツハイマー
病の有効な予防法や治療法は、見出されておらず、その
確立が要望されている。[0003] However, heretofore, no effective preventive or therapeutic method for this Alzheimer's disease has been found, and its establishment is demanded.
【０００４】[0004]
【発明が解決しようとする課題】本発明の課題は、アル
ツハイマー病の治療に有効な新規蛋白質である神経栄養
活性抑制物資を提供することである。SUMMARY OF THE INVENTION It is an object of the present invention to provide a novel protein which is effective for treating Alzheimer's disease and which is a neurotrophic activity inhibitor.
【０００５】[0005]
【課題を解決するための手段】本発明者は、アルツハイ
マー病患者の脳中成分を研究する過程で、正常人の脳中
には存在するが、アルツハイマー病患者の脳には存在し
なくなる新規な蛋白質であって、神経栄養活性抑制作用
があるものを見出し、この蛋白質を取り出すことに成功
した。In the course of studying the components in the brain of a patient with Alzheimer's disease, the present inventor has developed a novel novel compound that is present in the brain of a normal human but not present in the brain of a patient with Alzheimer's disease. They found a protein that has a neurotrophic activity inhibitory effect, and succeeded in extracting this protein.
【０００６】すなわち、この蛋白質は、ヒト脳組織より
抽出された抽出液をそのまま、又は、濃縮したのち、限
外ロ過、イオン交換クロマトグラフィー、ゲルろ過、高
速液体クロマトグラフィーなどの操作を組合せて、具体
的には、例えば、下記の実施例に示される手法により精
製、採取することができる。[0006] That is, this protein is obtained by concentrating an extract extracted from human brain tissue or directly concentrating the extract and then combining the operations such as ultrafiltration, ion exchange chromatography, gel filtration, and high performance liquid chromatography. Specifically, for example, it can be purified and collected by the method shown in the following example.
【０００７】かくして得られた本発明の新規タンパク質
は、以下の特性を有する。 分子量 ： 約５，０００（ＳＤＳ−ポリアクリルア
ミドゲル電気泳動法による） 性状 ： 白色無定形粉末 安定pH範囲： ３．０〜７．７ 熱安定性 ： ３７℃で２０時間保温又は１００℃で５
分間加熱しても神経栄養活性抑制作用を保持する。[0007] The novel protein of the present invention thus obtained has the following properties. Molecular weight: about 5,000 (by SDS-polyacrylamide gel electrophoresis) Properties: White amorphous powder Stable pH range: 3.0 to 7.7 Thermal stability: Insulation at 37 ° C for 20 hours or 100 ° C for 5 hours
It keeps the neurotrophic activity inhibitory effect even after heating for minutes.
【０００８】本発明の新規タンパク質の生理活性、すな
わち、神経栄養活性抑制作用は、下記試験例を示す方法
で測定した。[0008] The physiological activity of the novel protein of the present invention, ie, the effect of inhibiting neurotrophic activity, was measured by the method shown in the following Test Examples.
【０００９】さらに、この新規蛋白質の全アミノ酸配列
は、下記実施例に示す方法により決定した。その結果、
本発明は、下記の全アミノ酸配列を有することがわかっ
た。Further, the entire amino acid sequence of this novel protein was determined by the method shown in the following example. as a result,
The present invention was found to have the following entire amino acid sequence.
【００１０】 1 5 10 Met Asp Pro Glu Thr Cys Pro Cys Pro Ser 11 15 20 Gly Gly Ser Cys Thr Gys Ala Asp Ser Cys 21 25 30 Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys 31 35 40 Lys Lys Ser Cys Cys Ser Cys Cys Pro Ala 41 45 50 Glu Cys Glu Lys Cys Ala Lys Asp Cys Val 51 55 60 Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu 61 65 68 Ala Glu Lys Cys Ser Cys Cys Gln （配列表の配列番号１）1 5 10 Met Asp Pro Glu Thr Cys Pro Cys Pro Ser 11 15 20 Gly Gly Ser Cys Thr Gys Ala Asp Ser Cys 21 25 30 Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys 31 35 40 Lys Lys Ser Cys Cys Ser Cys Cys Pro Ala 41 45 50 Glu Cys Glu Lys Cys Ala Lys Asp Cys Val 51 55 60 Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu 61 65 68 Ala Glu Lys Cys Ser Cys Cys Gln (SEQ ID NO: 1 in Sequence Listing)
【００１１】下記試験例によっても示されるように、こ
の新規な蛋白質は、神経栄養活性抑制作用を示し、アル
ツハイマー病の診断、予防又は治療に用いて有効な物質
であることが判る。本発明者とその共同研究者は、さら
に、この新規な蛋白質の産生を司っている遺伝子を見出
し、これを用いて、遺伝子工学的な手法により、この新
規蛋白質を大量に生産し、これを用いることにより、ア
ルツハイマー病の診断、予防及び治療を行うことを考
え、鋭意研究を行った結果、その蛋白質をコードする遺
伝子（全長ｃＤＮＡ）を見出し、その核酸配列を決定す
ることに成功した。As shown in the following test examples, this novel protein exhibits a neurotrophic activity-suppressing action and is found to be an effective substance for use in diagnosis, prevention or treatment of Alzheimer's disease. The present inventors and their collaborators have further found a gene that is responsible for the production of this novel protein, and have used it to produce a large amount of this novel protein by genetic engineering techniques. By using it, the inventors considered the diagnosis, prevention and treatment of Alzheimer's disease and conducted intensive studies. As a result, they found a gene encoding the protein (full-length cDNA) and succeeded in determining its nucleic acid sequence.
【００１２】本発明の物質をコードする遺伝子の分離
と、その核酸配列の決定は、例えば、下記実施例に具体
的に示される方法によって行うことができる。The separation of the gene encoding the substance of the present invention and the determination of its nucleic acid sequence can be performed, for example, by the methods specifically shown in the following Examples.
【００１３】かくして決定された、ヒト脳中の神経栄養
活性抑制物質をコードするｃＤＮＡの核酸配列は次のと
おりであった。The thus determined nucleic acid sequence of the cDNA encoding the neurotrophic activity inhibitor in human brain was as follows.
【００１４】 ATG GAC CCT GAG ACC TGC CCC TGC CCT TCT GGT GGC TCC TGC ACC TGC 48 GCG GAC TCC TGC AAG TGC GAG GGA TGC AAA TGC ACC TCC TGC AAG AAG 96 AGC TGC TGC TCC TGC TGC CCT GCG GAG TGT GAG AAG TGT GCC AAG GAC 144 TGT GTG TGC AAA GGC GGA GAG GCA GCT GAG GCA GAA GCA GAG AAG TGC 192 AGC TGC TGC CAG 204 （配列表の配列番号２）ATG GAC CCT GAG ACC TGC CCC TGC CCT TCT GGT GGC TCC TGC ACC TGC 48 GCG GAC TCC TGC AAG TGC GAG GGA TGC AAA TGC ACC TCC TGC AAG AAG 96 AGC TGC TGC TCC TGC TGC CCT GCG GAG TGT GAG AAG TGT GCC AAG GAC 144 TGT GTG TGC AAA GGC GGA GAG GCA GCT GAG GCA GAA GCA GAG AAG TGC 192 AGC TGC TGC CAG 204 (SEQ ID NO: 2 in Sequence Listing)
【００１５】[0015]
【発明の効果】上記した新規蛋白質は、アルツハイマー
病の診断、治療に役立ち、さらには、上記した、この蛋
白質をコードするｃＤＮＡは、遺伝子工学的手法により
この蛋白質の大量生産を可能にする有用な遺伝子であ
る。また、この遺伝子は、アルツハイマー病の診断に有
効であり、さらには、直接的に遺伝子を導入することに
よりアルツハイマー病の治療に用いうることが期待され
る。The novel protein described above is useful for the diagnosis and treatment of Alzheimer's disease, and the cDNA encoding the protein is useful for mass production of this protein by genetic engineering techniques. Is a gene. In addition, this gene is effective for diagnosing Alzheimer's disease, and is expected to be usable for treatment of Alzheimer's disease by directly introducing the gene.
【００１６】以下、本発明を実施例に基づき、さらに詳
しく説明する。Hereinafter, the present invention will be described in more detail based on examples.
【００１７】実施例１ 本物質の分離、精製 正常ヒト大脳皮質の灰白質２０ｇに水６０mlを加え、ホ
モジナイズし、２０，０００ｇで１時間遠心した後、遠
心上清を５５mlを得た。Example 1 Separation and purification of this substance To 20 g of gray matter of normal human cerebral cortex was added 60 ml of water, homogenized, centrifuged at 20,000 g for 1 hour, and 55 ml of the centrifuged supernatant was obtained.
【００１８】得られた上清５５mlを、にアミコンＹＭ−
１０膜（商品名）を用いて限外ろ過し、分子量１０キロ
ダルトン以上の画分をＤＥＡＥ−セファセルカラム
（１．６cmφ×１６cm、ファルマシア社製）にのせ、洗
浄バッファー（５０mM ＮａＣｌ、２０mM Ｔｒｉｓ−
Ｃｌ（pH７．６））２００mlで洗浄後、５０mMから３０
０mM ＮａＣｌの直線濃度勾配をつけた２０mM Ｔｒｉ
ｓ−Ｃｌ（pH７．６）バッファー３２０mlで抽出した。
上記ＤＥＡＥ−セファセルカラムによるクロマトグラム
を図１に示す。フラクションNo.３１から３８までの抑
制活性を有する分画を集め（４０ml）、透析後フィコー
ル４００を用いて濃縮後ＴＳＫ Ｇ２０００ＳＷ（トー
ソー社製）でゲルろ過（カラムサイズ７．５mmφ×６c
m）し、フラクションNo.３０から３２の活性画分を集め
（２．５ml）、５mMリン酸バッファー（pH７．４）中で
透析した。上記ＴＳＫ Ｇ２０００ＳＷを用いたゲルろ
過クロマトグラフィーの結果を図２に示す。液量を５５
０μl まで濃縮後、Ｃ１８逆相ＨＰＬＣカラム（４．６
mmφ×２５cm、センシュー化学社製）にかけた。溶出に
は、０％から８０％アセトニトリルの直線勾配をつけた
５mMギ酸アンモニウム溶液を用いた。このＣ１８逆相Ｈ
ＰＬＣクロマトグラフィーの結果を図３に示す。図３に
示されるように、Ｃ１８逆相ＨＰＬＣクロマトグラフィ
ーによりシャープなピークが実質的に１つだけ得られ、
本発明の物質が単離されたことがわかる。55 ml of the obtained supernatant was added to Amicon YM-
Ultrafiltration using 10 membranes (trade name), the fraction having a molecular weight of 10 kilodalton or more was loaded on a DEAE-Sephacel column (1.6 cmφ × 16 cm, manufactured by Pharmacia), and washed with a washing buffer (50 mM NaCl, 20 mM Tris). −
Cl (pH 7.6)) after washing with 200 ml.
20 mM Tri with a linear gradient of 0 mM NaCl
Extracted with 320 ml of s-Cl (pH 7.6) buffer.
FIG. 1 shows a chromatogram obtained by the DEAE-Sephacel column. Fractions having inhibitory activities from fraction Nos. 31 to 38 were collected (40 ml), dialyzed, concentrated using Ficoll 400, and then subjected to gel filtration using TSK G2000SW (manufactured by Tosoh Corporation) (column size 7.5 mmφ × 6 c).
m), and the active fractions of fraction Nos. 30 to 32 were collected (2.5 ml) and dialyzed against 5 mM phosphate buffer (pH 7.4). FIG. 2 shows the results of gel filtration chromatography using the TSK G2000SW. 55
After concentration to 0 μl, a C18 reverse phase HPLC column (4.6
mmφ × 25 cm, manufactured by Senshu Chemical Co., Ltd.). For elution, a 5 mM ammonium formate solution with a linear gradient from 0% to 80% acetonitrile was used. This C18 reverse phase H
FIG. 3 shows the results of the PLC chromatography. As shown in FIG. 3, substantially only one sharp peak was obtained by C18 reverse phase HPLC chromatography,
It can be seen that the substance of the present invention was isolated.
【００１９】実施例２ 特性測定 実施例１で得られた物質について下記の種々の特性を
測定した。Example 2 Measurement of properties The following various properties were measured for the substance obtained in Example 1.
【００２０】 （１）紫外部吸収スペクトル 実施例１で得られた物質３μg の蒸留水溶液を用いて分
光光度計（ベックマン社製ＤＵ６５型）で紫外部吸収ス
ペクトルを測定した。結果を４図に示す。(1) Ultraviolet Absorption Spectrum An ultraviolet absorption spectrum was measured with a spectrophotometer (model DU65 manufactured by Beckman) using 3 μg of the distilled aqueous solution of the substance obtained in Example 1. The results are shown in FIG.
【００２１】 （２）安定性 実施例１で得られた物質を２０μg ／mlの水溶液に調製
し、その１０μl にトリフルオロ酢酸を最終濃度０．１
％となるように加え（pH３．０）、３７℃で２０時間加
温した後、凍結乾燥した。これを、ダルベコ社製リン酸
バッファー（ＰＢＳ（−））１０μl に溶解し、下記実
施例３に示す方法で抑制活性を測定したが活性の抑制活
性の減少は全く認められなかった。さらに、実施例１で
得た物質の２μg ／ml水溶液の１００μl をとり、３７
℃で２０時間又は１００℃で５分加熱した後、この溶液
の１０μl を用いて、前述と同様にして安定性試験を行
なったところ、全く抑制活性の減少が認められなかっ
た。(2) Stability The substance obtained in Example 1 was prepared in an aqueous solution of 20 μg / ml, and trifluoroacetic acid was added to 10 μl of the aqueous solution at a final concentration of 0.1 μl.
% (PH 3.0), heated at 37 ° C. for 20 hours, and lyophilized. This was dissolved in 10 µl of a phosphate buffer (PBS (-)) manufactured by Dalbeco and its inhibitory activity was measured by the method shown in Example 3 below, but no decrease in the inhibitory activity was observed. Further, 100 μl of a 2 μg / ml aqueous solution of the substance obtained in Example 1 was
After heating at 20 ° C. for 20 hours or 100 ° C. for 5 minutes, a stability test was carried out using 10 μl of this solution in the same manner as described above, and no decrease in inhibitory activity was observed.
【００２２】 （３）分子量 実施例１で得られた物質５μg を水１０μl に溶解し、
分子量マーカー（キモトリプシノーゲンＡ（分子量２，
５００）、チトクロムＣ（分子量１２，５００）、アプ
ロチニン（分子量６，５００）、バイオラッド社製）を
用いて、７．５％から２０％の濃度勾配のあるＳＤＳ−
ポリアクリルアミドゲル電気泳動で測定した結果、分子
量約５，０００ダルトンであることが確認された。この
電気泳動の結果を図５に示す。(3) Molecular weight 5 μg of the substance obtained in Example 1 was dissolved in 10 μl of water,
Molecular weight markers (chymotrypsinogen A (molecular weight 2,
500), cytochrome C (molecular weight: 12,500), aprotinin (molecular weight: 6,500), manufactured by Bio-Rad Co., Ltd.) and SDS- having a concentration gradient of 7.5% to 20%.
As a result of measurement by polyacrylamide gel electrophoresis, it was confirmed that the molecular weight was about 5,000 dalton. FIG. 5 shows the results of the electrophoresis.
【００２３】試験例 神経栄養活性抑制活性の測定 新生児ラットの大脳皮質より調製した細胞をゲラチン−
ポリオルイチンを塗布した６mmのミクロプレートに１．
７×１０4 個の細胞を撒き、実質例１と同様の方法で得
られたアルツハイマー病脳抽出液を１２５μg ／ml濃度
に調製した水溶液１００μl を含む無血清培地ＭＥＭＮ
２（イーグル基本培地にインシュリン、トランスフェリ
ン、プトレシン、プロゲステロン、亜セレン酸ナトリウ
ムを添加）に実施例１で得られた物質２０ngを加え、５
日間５％炭酸ガス培養槽中、３７℃で培養した。パラホ
ルムアルデヒドと９０％メタノール／５％酢酸溶液で固
定した後、マイクロチューブル結合タンパク２（ＭＡＰ
２）抗体（アマーシャム社製）を使ったＥＬＩＳＡでＭ
ＡＰ２量を定量した。一方、本物質を加えないでアルツ
ハイマー病脳抽出液を加えて培養した時のＭＡＰ２量を
定量し、ＭＡＰ２量が２％減少するかによって抑制活性
を表わした。Test Example Measurement of Neurotrophic Activity Inhibitory Activity Cells prepared from cerebral cortex of neonatal rat were cultured with gelatin-
1. Place a 6 mm microplate coated with polyoruitin.
A serum-free medium MEMN containing 100 μl of an aqueous solution prepared by dispersing 7 × 10 4 cells and preparing an Alzheimer's disease brain extract obtained in the same manner as in Example 1 to a concentration of 125 μg / ml.
2 (addition of insulin, transferrin, putrescine, progesterone, and sodium selenite to Eagle's basal medium) were added with 20 ng of the substance obtained in Example 1 and added.
The cells were cultured at 37 ° C. for 5 days in a 5% carbon dioxide gas culture tank. After fixation with paraformaldehyde and 90% methanol / 5% acetic acid solution, microtubule-binding protein 2 (MAP)
2) ELISA using antibody (Amersham)
The amount of AP2 was quantified. On the other hand, the amount of MAP2 when the Alzheimer's disease brain extract was added and cultured without adding this substance was quantified, and the inhibitory activity was indicated by whether the amount of MAP2 decreased by 2%.
【００２４】上記方法を用いて、本物質の量と抑制率と
の関係を測定した。結果を図６に示す。図６に示すよう
に、抑制活性は、本物質０．２μg ／mlで平衡となり、
その抑制活性は約９０％であった。Using the above method, the relationship between the amount of the substance and the inhibition rate was measured. FIG. 6 shows the results. As shown in FIG. 6, the inhibitory activity was equilibrated with 0.2 μg / ml of the substance,
Its inhibitory activity was about 90%.
【００２５】実施例３ アミノ酸配列の分析 実施例１で得られた物質２００μg を、常法によりピリ
ジルエチル化た。ピリジルエチル化した本物質５０μg
を常法により臭化シアン分解した。ピリジルエチル化し
た本物質を０．１M Ｔｒｉｓ−Ｃｌ（pH８．０）溶液１
００μl に溶解し、ＴＲＣＫ−トリプシン（シグマ社
製）または endoproteinase Ａｓｐ−Ｎ（ベーリンガー
社製）または S. aureus Ｖ８ protease （シグマ社
製）０．５μg を加え、３７℃、５時間反応させた。以
上の４種類の方法を用いて得られたペプチド断片をそれ
ぞれＣ１８逆相ＨＰＬＣ（０〜８０％アセトニトリル／
０．１％トリフルオロ酢酸溶液）で分離し、タンパクシ
ークエンサー（アプライドバイオシステム社４７７Ａ
型）にかけて分析し、得られたピークの保持時間と標準
物質のそれを比較解読して本物質の全アミノ酸配列を決
定した。その結果、本物質は下記の全アミノ酸配列を有
することがわかった。 1 5 10 Met Asp Pro Glu Thr Cys Pro Cys Pro Ser 11 15 20 Gly Gly Ser Cys Thr Cys Ala Asp Ser Cys 21 25 30 Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys 31 35 40 Lys Lys Ser Cys Cys Ser Cys Cys Pro Ala 41 45 50 Glu Cys Glu Lys Cys Ala Lys Asp Cys Val 51 55 60 Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu 61 65 68 Ala Glu Lys Cys Ser Cys Cys GlnExample 3 Analysis of Amino Acid Sequence 200 μg of the substance obtained in Example 1 was subjected to pyridylethylation by a conventional method. Pyridylethylated substance 50μg
Was decomposed with cyanogen bromide by a conventional method. Pyridylethylated substance is 0.1 M Tris-Cl (pH 8.0) solution 1
After dissolving in 00 μl, 0.5 μg of TRCK-trypsin (Sigma), endoproteinase Asp-N (Boehringer) or S. aureus V8 protease (Sigma) was added, and the mixture was reacted at 37 ° C. for 5 hours. Each of the peptide fragments obtained by using the above four methods was subjected to C18 reverse phase HPLC (0 to 80% acetonitrile /
Separation with a 0.1% trifluoroacetic acid solution) and a protein sequencer (477A, Applied Biosystems)
), The retention time of the obtained peak and that of the standard substance were compared and decoded to determine the entire amino acid sequence of the substance. As a result, this substance was found to have the following entire amino acid sequence. 1 5 10 Met Asp Pro Glu Thr Cys Pro Cys Pro Ser 11 15 20 Gly Gly Ser Cys Thr Cys Ala Asp Ser Cys 21 25 30 Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys 31 35 40 Lys Lys Ser Cys Cys Ser Cys Cys Pro Ala 41 45 50 Glu Cys Glu Lys Cys Ala Lys Asp Cys Val 51 55 60 Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu 61 65 68 Ala Glu Lys Cys Ser Cys Cys Gln
【００２６】参考例１ 本遺伝子の分離 ヒト脳から抽出された神経栄養活性抑制物質のアミノ酸
配列をもとに5′ATGGATCCCGAGACCTGCCC, 5′CTGGCAGCAG
CTGCACTTCTC の２つのオリゴヌクレオチドを合成しこれ
らをプライマーとして、ヒト大脳より調製したメッセン
ジャーＲＮＡを鋳型にして、逆転写酵素によりｃＤＮＡ
とした後、ポリメレース連鎖反応を行いその反応生成物
をプラスミドベクターpUC １９にサブクローン化し、そ
の塩基配列を決定し、これが神経栄養活性抑制物質のア
ミノ酸配列と対応することを確認した。Reference Example 1 Isolation of the present gene 5'ATGGATCCCGAGACCTGCCC, 5'CTGGCAGCAG based on the amino acid sequence of a neurotrophic activity inhibitor extracted from human brain
Two oligonucleotides of CTGCACTTCTC were synthesized, and these were used as primers, using messenger RNA prepared from human cerebrum as a template, and cDNA using reverse transcriptase.
After that, a polymerase chain reaction was performed, and the reaction product was subcloned into a plasmid vector pUC19, and its nucleotide sequence was determined. It was confirmed that the nucleotide sequence corresponded to the amino acid sequence of a neurotrophic activity inhibitor.
【００２７】正常ヒト大脳のメッセンジャーＲＮＡに対
して作られたｃＤＮＡライブラリーを用いて、１×１０
6 個のクローンをプレートに生育させ、ニトロセルロー
ス膜に転写し、上記のサブクローン化された核酸配列を
32Ｐ でラベルしてプロープを作製しこれを５０％ホル
ムアミド、５ＸＳＳＣ（0.15M Nacl, 15mMクエン酸ナト
リウム、pH7.0 ）を含むハイプリダイゼーション液中で
４２℃にて１８時間、上記ニトロセルロース膜にハイブ
リッド形成させ、その後フィルターを洗浄し最終的に５
５℃で０．１Ｘ ＳＳＣ（0.15M NaCl, 0.15mMクエン酸
ナトリウム、pH7.0 ）オートラジオグラフィーを行い上
記プロープに対して特異的なｃＤＮＡを２４個単離し
た。このｃＤＮＡの塩基配列を決定したところ６８個の
アミノ酸をコードする核酸配列の存在が見出された。Using a cDNA library prepared for messenger RNA of normal human cerebrum, 1 × 10
Six clones are grown on a plate, transferred to nitrocellulose membrane, and the subcloned nucleic acid sequence
A probe was prepared by labeling with 32 P, and the probe was placed in a hybridization solution containing 50% formamide and 5 × SSC (0.15 M Nacl, 15 mM sodium citrate, pH 7.0) at 42 ° C. for 18 hours for 18 hours. And then wash the filter and finally 5
Autoradiography was performed at 5 ° C. with 0.1 × SSC (0.15 M NaCl, 0.15 mM sodium citrate, pH 7.0) to isolate 24 cDNAs specific to the probe. When the nucleotide sequence of this cDNA was determined, the presence of a nucleic acid sequence encoding 68 amino acids was found.
【００２８】参考例２ それぞれメッセンジャーＲＮＡを抽出し、その２マイク
ログラムを変性アガロースゲル中で電気泳動を行い、そ
の後ニトロセルロース膜に転写し、上記ｃＤＮＡをプロ
ーブとして上記と同様のハイブリダイゼーション液中で
４２℃で１８時間ハイブリッド形成を行い、その後フィ
ルターを洗浄し、最終的に０．１Ｘ ＳＳＣ，０．１％
ＳＤＳ中で６５℃で洗浄し、オートラジオグラフィーを
行った。その結果アルツハイマー病、正常脳において、
ともに約５００ｂｐの大きさのメッセンジャーＲＮＡが
認められ、アルツハイマー病においてこのメッセンジャ
ーＲＮＡの量が減少していることが見いだされた。Reference Example 2 Each messenger RNA was extracted, and 2 micrograms thereof were subjected to electrophoresis in a denaturing agarose gel, then transferred to a nitrocellulose membrane, and the above cDNA was used as a probe in the same hybridization solution as described above. Hybridization was carried out at 42 ° C. for 18 hours, after which the filters were washed and finally 0.1 × SSC, 0.1%
After washing in SDS at 65 ° C., autoradiography was performed. As a result, in Alzheimer's disease, normal brain,
In both cases, a messenger RNA having a size of about 500 bp was observed, and it was found that the amount of this messenger RNA was reduced in Alzheimer's disease.
【００２９】 配列番号：１ 配列の長さ：６８ 配列の型：アミノ酸 トポロジー：直鎖状（ただし、高次構造は直鎖ではない
複雑な構造をしている） 配列の種類：ペプチド（蛋白） 起源：ヒト脳組織抽出物 配列： 1 5 10 Met Asp Pro Glu Thr Cys Pro Cys Pro Ser 11 15 20 Gly Gly Ser Cys Thr Cys Ala Asp Ser Cys 21 25 30 Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys 31 35 40 Lys Lys Ser Cys Cys Ser Cys Cys Pro Ala 41 45 50 Glu Cys Glu Lys Cys Ala Lys Asp Cys Val 51 55 60 Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu 61 65 68 Ala Glu Lys Cys Ser Cys Cys GlnSEQ ID NO: 1 Sequence length: 68 Sequence type: amino acid Topology: linear (however, the higher-order structure has a complex structure that is not linear) Sequence type: peptide (protein) Origin: Human brain tissue extract Sequence: 1 5 10 Met Asp Pro Glu Thr Cys Pro Cys Pro Ser 11 15 20 Gly Gly Ser Cys Thr Cys Ala Asp Ser Cys 21 25 30 Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys 31 35 40 Lys Lys Ser Cys Cys Cys Ser Cys Cys Pro Ala 41 45 50 50 Glu Cys Glu Lys Cys Ala Lys Asp Cys Val 51 55 60 Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu 61 65 68 Ala Glu Lys Cys Ser Cys Cys Gln
【００３０】 配列番号：２ 配列の長さ：２０４ 配列の型：核酸 鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：ｃＤＮＡ ｔｏ ｍｅｓｓｅｎｇｅｒ Ｒ
ＮＡ 起源：ヒト大脳 配列： ATG GAC CCT GAG ACC TGC CCC TGC CCT TCT GGT GGC TCC TGC ACC TGC 48 GCG GAC TCC TGC AAG TGC GAG GGA TGC AAA TGC ACC TCC TGC AAG AAG 96 AGC TGC TGC TCC TGC TGC CCT GCG GAG TGT GAG AAG TGT GCC AAG GAC 144 TGT GTG TGC AAA GGC GGA GAG GCA GCT GAG GCA GAA GCA GAG AAG TGC 192 AGC TGC TGC CAG 204SEQ ID NO: 2 Sequence length: 204 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: cDNA to messenger R
NA Origin: Human cerebrum Sequence: ATG GAC CCT GAG ACC TGC CCC TGC CCT TCT GGT GGC TCC TGC ACC TGC 48 GCG GAC TCC TGC AAG TGC GAG GGA TGC AAA TGC ACC TCC TGC AGC AAG 96 AGC TGC TGC TCC TGC TGC CCT GCG GAG TGT GAG AAG TGT GCC AAG GAC 144 TGT GTG TGC AAA GGC GGA GAG GCA GCT GAG GCA GAA GCA GAG AAG TGC 192 AGC TGC TGC CAG 204
【００３１】[0031]
【図１】正常ヒト大脳皮質をホモジナイズして限外ろ過
し、分子量１０キロダルトン以上の画分をＤＥＡＥ−セ
ファセルカラムにかけたクロマトグラムである。FIG. 1 is a chromatogram obtained by homogenizing a normal human cerebral cortex, performing ultrafiltration, and applying a fraction having a molecular weight of 10 kDa or more to a DEAE-Sephacel column.
【図２】本発明の物質（蛋白質）の精製過程において、
抑制活性を有するフラクションをゲルろ過したクロマト
グラムである。FIG. 2 shows the purification process of the substance (protein) of the present invention.
It is the chromatogram which carried out the gel filtration of the fraction which has inhibitory activity.
【図３】本発明の物質をＣ１８逆相ＨＰＬＣにかけたク
ロマトグラムである。FIG. 3 is a chromatogram of a substance of the present invention subjected to C18 reverse phase HPLC.
【図４】本発明の物質の紫外部吸収スペクトル図であ
る。FIG. 4 is an ultraviolet absorption spectrum of the substance of the present invention.
【図５】本発明の物質のＳＤＳ−ＰＡＧＥの泳動パター
ンを示す図である。FIG. 5 is a view showing an SDS-PAGE electrophoresis pattern of the substance of the present invention.
【図６】本発明の物質の量と抑制率との関係を示す図で
ある。FIG. 6 is a graph showing the relationship between the amount of the substance of the present invention and the suppression rate.
【図７】ヒト神経栄養活性抑制物質ｃＤＮＡをプローブ
としたノーザン解析の結果を示す。アルツハイマー病に
おいて神経栄養活性抑制物質のメッセンジャーＲＮＡ量
が減少していることを示す、クロマトグラムである。FIG. 7 shows the results of Northern analysis using a human neurotrophic activity inhibitor cDNA as a probe. 1 is a chromatogram showing that the amount of messenger RNA of a neurotrophic activity inhibitor is reduced in Alzheimer's disease.
Claims (2)
る塩基配列を有するＤＮＡ。[Claim 1] Met Asp Pro Glu Thr Cys Pro Cys Pro Ser Gly Gly Ser Cys Thr Cys Ala Asp Ser Cys Lys Cys Glu Gly Cys Lys Cys Thr Ser Cys Lys Lys Ser Cys Cys Ser Cys Cys Pro Ala Glu Cys Glu Lys Cys Ala Lys Asp Cys Val Cys Lys Gly Gly Glu Ala Ala Glu Ala Glu Ala Glu Lys Cys Ser Cys DNA having a base sequence encoding a protein having an amino acid sequence represented by Cys Gln.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/696,051 US5214031A (en) | 1990-05-09 | 1991-05-06 | Growth-inhibitory factor obtained from human brain |
| DE69121964T DE69121964T2 (en) | 1990-05-09 | 1991-05-07 | Growth-inhibiting factor and cDNA coding for the growth-inhibiting factor |
| AT91401221T ATE142643T1 (en) | 1990-05-09 | 1991-05-07 | GROWTH INHIBITING FACTOR AND CDNS CODING FOR GROWTH INHIBITING FACTOR |
| EP91401221A EP0458673B1 (en) | 1990-05-09 | 1991-05-07 | Growth-inhibitory factor and cDNA coding for growth inhibitory factor |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP27937190 | 1990-10-19 | ||
| JP2-279371 | 1990-10-19 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04169600A JPH04169600A (en) | 1992-06-17 |
| JP2904935B2 true JP2904935B2 (en) | 1999-06-14 |
Family
ID=17610222
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2410164A Expired - Fee Related JP2904935B2 (en) | 1990-05-09 | 1990-12-13 | Neurotrophic activity inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2904935B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5534615A (en) * | 1994-04-25 | 1996-07-09 | Genentech, Inc. | Cardiac hypertrophy factor and uses therefor |
| WO1995029237A1 (en) * | 1994-04-25 | 1995-11-02 | Genentech, Inc. | Cardiotrophin and uses therefor |
| US7258983B2 (en) | 1994-04-25 | 2007-08-21 | Genentech, Inc. | Cardiotrophin-1 compositions and methods for the treatment of tumor |
| US6472585B1 (en) | 1994-04-25 | 2002-10-29 | Genentech, Inc. | Cardiotrophin-1 defective mouse |
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1990
- 1990-12-13 JP JP2410164A patent/JP2904935B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04169600A (en) | 1992-06-17 |
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