JP6339399B2 - Ovarian clear cell adenocarcinoma cell detection method, ovarian clear cell adenocarcinoma diagnostic agent, ovarian clear cell adenocarcinoma diagnostic primer, ovarian clear cell adenocarcinoma diagnostic kit, and ovarian clear cell adenocarcinoma diagnostic probe - Google Patents
Ovarian clear cell adenocarcinoma cell detection method, ovarian clear cell adenocarcinoma diagnostic agent, ovarian clear cell adenocarcinoma diagnostic primer, ovarian clear cell adenocarcinoma diagnostic kit, and ovarian clear cell adenocarcinoma diagnostic probe Download PDFInfo
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Description
本発明は、卵巣明細胞腺癌細胞の検出方法、卵巣明細胞腺癌診断薬、卵巣明細胞腺癌診断用プライマー、卵巣明細胞腺癌診断用キット、及び卵巣明細胞腺癌診断用プローブに関する。 The present invention relates to a method for detecting ovarian clear cell adenocarcinoma cells, a diagnostic agent for ovarian clear cell adenocarcinoma, a primer for ovarian clear cell adenocarcinoma diagnosis, a kit for diagnosing ovarian clear cell adenocarcinoma, and a probe for diagnosing ovarian clear cell adenocarcinoma. .
卵巣癌は、その解剖学的位置関係から、画像による早期診断は困難なことが多い。また、卵巣発癌機構の解析も大幅に遅れており、分子標的薬を含む新規治療法の開発へも深刻な影響を及ぼしている。最近、一部の卵巣癌（明細胞腺癌・類内膜腺癌）の前駆病変として、子宮内膜症性卵巣嚢胞が注目されている。これは、月経を有する約10％の女性に認められる現代病で、治療法として、40歳代で6cm以上、閉経後は大きさによらず全て摘出することが推奨されている。しかしながら、手術侵襲や術後後遺症等のリスクを考慮すると、癌合併を予知する早期診断法の確立が必要である。
卵巣癌は、Clear Cell（明細胞）、Endometrioid（類内膜性）、Serous（漿液性）、Mucinous（粘液性）の４種の組織型に分類することができる。なかでも卵巣明細胞腺癌は、既存の抗癌剤には低感受性で治療に苦渋することが多い点から、新規治療法の開発が望まれている。これまでに、明細胞腺癌に特異的に発現する遺伝子としてHepatocyte nuclear factor (HNF)-1βが報告されている（非特許文献１）。しかし、卵巣明細胞腺癌症例の中にはHNF-1β発現の陰性例も認められる。したがって、より正確な診断を実現するためにも、新規診断マーカーの開発が望まれている。
Ovarian cancer is often difficult to diagnose early using images because of its anatomical positional relationship. In addition, the analysis of ovarian carcinogenesis is greatly delayed, and it has a serious impact on the development of new therapies including molecular targeted drugs. Recently, endometriotic ovarian cysts have attracted attention as precursors of some ovarian cancers (clear cell adenocarcinoma, endometrioid adenocarcinoma). This is a modern disease found in about 10% of women with menstruation. As a treatment, it is recommended to remove 6 cm or more in the 40s and after menopause regardless of size. However, considering risks such as surgical invasion and postoperative sequelae, it is necessary to establish an early diagnosis method for predicting cancer complications.
Ovarian cancer can be classified into four tissue types: Clear Cell (clear cell), Endometrioid (endometrioid), Serous (serous), and Mucinous (mucous). In particular, ovarian clear cell adenocarcinoma is less sensitive to existing anticancer agents and is often difficult to treat. Therefore, development of a new treatment method is desired. So far, Hepatocyte nuclear factor (HNF) -1β has been reported as a gene specifically expressed in clear cell adenocarcinoma (Non-patent Document 1). However, some cases of ovarian clear cell adenocarcinoma are negative for HNF-1β expression. Therefore, development of a new diagnostic marker is desired in order to realize a more accurate diagnosis.
Left right determinant factor (LEFTY)はtransforming growth factor β（TGF-β）スーパーファミリーの１因子であり、中内胚葉の形成や胚形成における左右軸決定に重要な役割を担う分子として同定された（非特許文献２〜３参照）。他のTGF-βファミリー分子と異なり、同じスーパーファミリー因子であるNodalと拮抗性に働くことが知られており、LEFTYとNodalの活性のバランスにより適切な発生が行われている（非特許文献４参照）。また、ES細胞は他の幹細胞に比べLEFTY遺伝子のメチル化が少なく、エピジェネティックにNodal/TGF-β-LEFTYシグナルが調節されていることが知られている（非特許文献５〜６参照）。さらに、miR-302により制御されていることも明らかとなり、microRNA依存LEFTY発現はES細胞において、細胞の多機能性と分化性の均衡を保っていると考えられている（非特許文献７参照）。
近年、腫瘍におけるLEFTYの発現は、腫瘍増殖抑制因子として機能していることが報告され、ヌードマウスを用いたin vivo実験においても、腫瘍細胞にLEFTYを導入した結果増殖が制御された（非特許文献８〜１０参照）。しかし、これまでに、LEFTYと卵巣明細胞腺癌との関連は知られていない。
Left right determinant factor (LEFTY) is a member of the transforming growth factor β (TGF-β) superfamily and has been identified as a molecule that plays an important role in determining the left-right axis in mesendoderm formation and embryogenesis (non- (See Patent Documents 2-3). Unlike other TGF-β family molecules, it is known to act antagonistically with Nodal, which is the same superfamily factor, and is appropriately generated by the balance of the activities of LEFTY and Nodal (Non-patent Document 4). reference). In addition, ES cells are known to have less methylation of LEFTY gene than other stem cells and epigenetic regulation of Nodal / TGF-β-LEFTY signal (see Non-Patent Documents 5 to 6). Furthermore, it is also clarified that it is controlled by miR-302, and microRNA-dependent LEFTY expression is considered to maintain a balance between the multifunctionality and differentiation of cells in ES cells (see Non-Patent Document 7). .
Recently, it has been reported that the expression of LEFTY in tumors functions as a tumor growth inhibitory factor, and in vivo experiments using nude mice, the growth was controlled as a result of introducing LEFTY into tumor cells (non-patented). Reference 8-10). However, to date, the association between LEFTY and ovarian clear cell adenocarcinoma is unknown.
本発明は上記事情に鑑みてなされたものであり、卵巣明細胞腺癌細胞を容易に検出可能とする、卵巣明細胞腺癌細胞の検出方法、卵巣明細胞腺癌診断薬、卵巣明細胞腺癌診断用プライマー、卵巣明細胞腺癌診断用キット、及び卵巣明細胞腺癌診断用プローブの提供を課題とする。 The present invention has been made in view of the above circumstances, and can detect ovarian clear cell adenocarcinoma cells easily, a method for detecting ovarian clear cell adenocarcinoma cells, an ovarian clear cell adenocarcinoma diagnostic agent, an ovarian clear cell gland An object is to provide a primer for diagnosing cancer, a kit for diagnosing ovarian clear cell adenocarcinoma, and a probe for diagnosing ovarian clear cell adenocarcinoma.
本発明者らは、上記課題を解決すべく鋭意研究した結果、卵巣明細胞腺癌の病原部でLeft right determinant factor（LEFTY）が特異的に発現することを見出し、この分子を標的とすることで、卵巣明細胞腺癌細胞の検出が可能となることを見出した。 As a result of diligent research to solve the above problems, the present inventors have found that left right determinant factor (LEFTY) is specifically expressed in the pathogenic part of ovarian clear cell adenocarcinoma and targeting this molecule. And found that ovarian clear cell adenocarcinoma cells can be detected.
すなわち、本発明は、下記の特徴を有する卵巣明細胞腺癌細胞の検出方法、卵巣明細胞腺癌診断薬、卵巣明細胞腺癌診断用プライマー、卵巣明細胞腺癌診断用キット、及び卵巣明細胞腺癌診断用プローブを提供するものである。 That is, the present invention provides a method for detecting ovarian clear cell adenocarcinoma cells having the following characteristics, an ovarian clear cell adenocarcinoma diagnostic agent, an ovarian clear cell adenocarcinoma diagnostic primer, an ovarian clear cell adenocarcinoma diagnostic kit, and an ovarian clear cell A probe for diagnosing cell adenocarcinoma is provided.
（１）生体試料中の卵巣明細胞腺癌細胞の検出方法であって、卵巣明細胞腺癌細胞の検出対象者から採取された卵巣組織の病理検体に対して、以下の（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、以下の（ａ）〜（ｂ）のいずれかのアミノ酸配列の部分配列を有するタンパク質、又は、以下の（ｃ）のアミノ酸配列のうち配列番号１若しくは２で表されるアミノ酸配列に対して、同一性を有するアミノ酸配列の部分配列を有するタンパク質を腫瘍マーカーとして用いる工程Ａを有することを特徴とする卵巣明細胞腺癌細胞の検出方法。
（ａ）配列番号１又は２で表されるアミノ酸配列、
（ｂ）配列番号１又は２で表されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換、又は付加されているアミノ酸配列、
（ｃ）配列番号１又は２で表されるアミノ酸配列に対して、８０％以上の同一性を有するアミノ酸配列
（２）前記工程Ａは、前記卵巣組織の病理検体中の前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、前記（ａ）〜（ｂ）のいずれかのアミノ酸配列の部分配列を有するタンパク質、又は、以下の（ｃ）のアミノ酸配列のうち配列番号１若しくは２で表されるアミノ酸配列に対して、同一性を有するアミノ酸配列の部分配列を有するタンパク質を検出する工程Ａ１を有する前記（１）に記載の卵巣明細胞腺癌細胞の検出方法。
（３）前記工程Ａ１は、前記タンパク質に対する特異的抗体を用いて前記タンパク質を検出する工程である前記（２）に記載の卵巣明細胞腺癌細胞の検出方法。
（４）前記工程Ａは、前記卵巣組織の病理検体中の前記タンパク質を規定するポリヌクレオチドの発現を検出する工程Ａ２を有する前記（１）に記載の卵巣明細胞腺癌細胞の検出方法。
（５）前記工程Ａ２は、以下の（ｄ）〜（ｆ）のいずれかの塩基配列に基づいて作製したプローブ及び／又はプライマーを用いて、前記タンパク質を規定するポリヌクレオチドの発現を検出する工程である前記（４）に記載の卵巣明細胞腺癌細胞の検出方法。
（ｄ）配列番号３又は４で表される塩基配列、
（ｅ）配列番号３又は４で表される塩基配列において、１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列、
（ｆ）配列番号３又は４で表される塩基配列に対して、８０％以上の同一性を有する塩基配列
（６）前記工程Ａ２は、配列番号５で表される塩基配列、及び／又は、配列番号６で表される塩基配列を有するプローブ及び／又はプライマーを用いて前記タンパク質を規定するポリヌクレオチドの発現を検出する工程である前記（４）又は（５）に記載の卵巣明細胞腺癌細胞の検出方法。
(1) A method for detecting ovarian clear cell adenocarcinoma cells in a biological sample , comprising the following (a) to (a) to a pathological specimen of ovarian tissue collected from a subject to be detected of ovarian clear cell adenocarcinoma cells : protein having an amino acid sequence of any of c), the following (a) a protein having a partial sequence of the amino acid sequence of any of ~ (b), or, or SEQ ID NO: 1 of the amino acid sequence of the following (c) A method for detecting ovarian clear cell adenocarcinoma cells, comprising a step A using a protein having a partial sequence of an amino acid sequence having identity to the amino acid sequence represented by 2, as a tumor marker.
(A) the amino acid sequence represented by SEQ ID NO: 1 or 2,
(B) in the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence in which one to several amino acids are deleted, substituted, or added;
(C) Amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 2 (2) The step A comprises the steps (a) to (a) in the pathological specimen of the ovarian tissue protein having an amino acid sequence of any of c), prior to Symbol (a) a protein having a partial sequence of the amino acid sequence of any of ~ (b), or, or SEQ ID NO: 1 of the amino acid sequence of the following (c) The method for detecting ovarian clear cell adenocarcinoma cells according to (1) above, comprising a step A1 of detecting a protein having a partial sequence of an amino acid sequence having identity to the amino acid sequence represented by 2 .
(3) The method for detecting ovarian clear cell adenocarcinoma cells according to (2), wherein the step A1 is a step of detecting the protein using a specific antibody against the protein.
(4) The method for detecting ovarian clear cell adenocarcinoma cells according to (1), wherein the step A includes a step A2 of detecting expression of a polynucleotide defining the protein in the pathological specimen of the ovarian tissue .
(5) The step A2 is a step of detecting the expression of a polynucleotide defining the protein using a probe and / or primer prepared based on any one of the following base sequences (d) to (f): The method for detecting ovarian clear cell adenocarcinoma cells according to (4) above.
(D) the base sequence represented by SEQ ID NO: 3 or 4,
(E) a nucleotide sequence in which one to several nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 3 or 4;
(F) a base sequence having 80% or more identity to the base sequence represented by SEQ ID NO: 3 or 4 (6) the step A2 comprises the base sequence represented by SEQ ID NO: 5 and / or Ovarian clear cell adenocarcinoma according to (4) or (5) above, which is a step of detecting the expression of a polynucleotide defining the protein using a probe and / or primer having the base sequence represented by SEQ ID NO: 6 Cell detection method .
（７）卵巣明細胞腺癌細胞の検出対象者から採取された卵巣組織の病理検体に対して用いられ、以下の（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、以下の（ａ）〜（ｂ）のいずれかのアミノ酸配列の部分配列を有するタンパク質、又は、以下の（ｃ）のアミノ酸配列のうち配列番号１若しくは２で表されるアミノ酸配列に対して、同一性を有するアミノ酸配列の部分配列を有するタンパク質に対する特異的抗体を含有することを特徴とする卵巣明細胞腺癌診断薬。
（ａ）配列番号１又は２で表されるアミノ酸配列、
（ｂ）配列番号１又は２で表されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換、又は付加されているアミノ酸配列、
（ｃ）配列番号１又は２で表されるアミノ酸配列に対して、８０％以上の同一性を有するアミノ酸配列
（８）卵巣明細胞腺癌細胞の検出対象者から採取された卵巣組織の病理検体に対して用いられ、配列番号５で表される塩基配列を有するプライマー、及び配列番号６で表される塩基配列を有するプライマーを含むことを特徴とする卵巣明細胞腺癌診断用プライマー。
（９）前記（８）に記載の卵巣明細胞腺癌診断用プライマーを含むことを特徴とする卵巣明細胞腺癌診断用キット。
（１０）卵巣明細胞腺癌細胞の検出対象者から採取された卵巣組織の病理検体に対して用いられ、以下の（ｇ）〜（ｉ）のいずれかを含むことを特徴とする卵巣明細胞腺癌診断用プローブ。
（ｇ）配列番号７又は８で表される塩基配列の少なくとも一部を含む塩基配列、又は配列番号７又は８で表される塩基配列と相補的な塩基配列の少なくとも一部を含む塩基配列、を含むヌクレオチド断片、
（ｈ）配列番号７又は８で表される塩基配列において１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列の少なくとも一部を含む塩基配列、又は配列番号７又は８で表される塩基配列において１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列と相補的な塩基配列の少なくとも一部を含む塩基配列、を含むヌクレオチド断片、
（ｉ）配列番号７又は８で表される塩基配列の少なくとも一部を含む塩基配列、又は配列番号７又は８で表される塩基配列と相補的な塩基配列の少なくとも一部を含む塩基配列を有するポリヌクレオチドと、ストリンジェントな条件下でハイブリダイズするポリヌクレオチド又はヌクレオチド断片
（１１）前記（１０）に記載の卵巣明細胞腺癌診断用プローブを含むことを特徴とする卵
巣明細胞腺癌診断用キット。
（１２）卵巣明細胞腺癌細胞の検出対象者から採取された卵巣組織の病理検体に対して用いられ、以下の（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、以下の（ａ）〜（ｂ）のいずれかのアミノ酸配列の部分配列を有するタンパク質、又は、以下の（ｃ）のアミノ酸配列のうち配列番号１若しくは２で表されるアミノ酸配列に対して、同一性を有するアミノ酸配列の部分配列を有するタンパク質に対する特異的抗体を含有することを特徴とする卵巣明細胞腺癌診断キット。
（ａ）配列番号１又は２で表されるアミノ酸配列、
（ｂ）配列番号１又は２で表されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換、又は付加されているアミノ酸配列、
（ｃ）配列番号１又は２で表されるアミノ酸配列に対して、８０％以上の同一性を有するアミノ酸配列
( 7 ) A protein having any one of the following amino acid sequences (a) to (c), which is used for a pathological specimen of ovarian tissue collected from a detection subject of ovarian clear cell adenocarcinoma cells, a) a protein having a partial sequence of the amino acid sequence of any one of ( b ) or the amino acid sequence represented by SEQ ID NO: 1 or 2 of the following amino acid sequences (c) A diagnostic agent for ovarian clear cell adenocarcinoma comprising a specific antibody against a protein having a partial sequence of an amino acid sequence .
(A) the amino acid sequence represented by SEQ ID NO: 1 or 2,
(B) in the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence in which one to several amino acids are deleted, substituted, or added;
(C) Amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 2 ( 8 ) Pathological specimen of ovarian tissue collected from a subject to detect ovarian clear cell adenocarcinoma cells And a primer having a base sequence represented by SEQ ID NO: 5 and a primer having a base sequence represented by SEQ ID NO: 6.
( 9 ) A kit for diagnosing ovarian clear cell adenocarcinoma comprising the primer for diagnosing ovarian clear cell adenocarcinoma according to ( 8 ) above.
( 10 ) Ovarian clear cell, which is used for a pathological specimen of ovarian tissue collected from a subject to detect ovarian clear cell adenocarcinoma cells and contains any of the following (g) to (i): Adenocarcinoma diagnostic probe.
(G) a base sequence comprising at least a part of the base sequence represented by SEQ ID NO: 7 or 8, or a base sequence comprising at least a part of the base sequence complementary to the base sequence represented by SEQ ID NO: 7 or 8. A nucleotide fragment comprising
(H) a nucleotide sequence comprising at least a part of a nucleotide sequence in which one to several nucleotides are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 7 or 8, or SEQ ID NO: 7 or 8 A nucleotide fragment comprising a base sequence comprising at least a part of a base sequence complementary to a base sequence in which 1 to several nucleotides are deleted, substituted or added in the represented base sequence,
(I) a base sequence comprising at least a part of the base sequence represented by SEQ ID NO: 7 or 8, or a base sequence comprising at least a part of the base sequence complementary to the base sequence represented by SEQ ID NO: 7 or 8 A diagnosis of ovarian clear cell adenocarcinoma comprising the polynucleotide or nucleotide fragment that hybridizes under stringent conditions ( 11 ) and the probe for ovarian clear cell adenocarcinoma according to ( 10 ) above For kit.
( 12 ) A protein having any one of the following amino acid sequences (a) to (c), which is used for a pathological specimen of ovarian tissue collected from a subject to detect ovarian clear cell adenocarcinoma cells, a) a protein having a partial sequence of the amino acid sequence of any one of ( b ) or the amino acid sequence represented by SEQ ID NO: 1 or 2 of the following amino acid sequences (c) A diagnostic kit for ovarian clear cell adenocarcinoma comprising a specific antibody against a protein having a partial sequence of an amino acid sequence .
(A) the amino acid sequence represented by SEQ ID NO: 1 or 2,
(B) in the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence in which one to several amino acids are deleted, substituted, or added;
(C) an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 2
本発明によれば、卵巣明細胞腺癌細胞を検出することが可能であるので、卵巣明細胞腺癌を早期に発見できる。 According to the present invention, since ovarian clear cell adenocarcinoma cells can be detected, ovarian clear cell adenocarcinoma can be discovered early.
＜Ｌｅｆｔ ｒｉｇｈｔ ｄｅｔｅｒｍｉｎａｎｔ ｆａｃｔｏｒ＞
プロテオミクス法は、タンパク質を二次元電気泳動法で展開し、質量分析により新規バイオマーカーを網羅的に探索する手法である。しかし、その解析には、凍結試料の状態により様々な制限があった。
発明者らはまず、卵巣明細胞腺癌患者由来の卵巣明細胞腺癌組織のホルマリン固定パラフィン包埋（FFPE）材料を得て、マイクロダイセクションにより明細胞腺癌細胞を含む病変部の切片を切り出した。次いで、病変部からタンパク質を抽出し、予めタンパク質をトリプシンで分解し、直接質量分析で測定・同定するショットガンプロテオミクス法を行った。これら一連の方法により病変部でのタンパク質の発現を効率よく解析することができ、卵巣明細胞腺癌細胞において高発現するタンパク質であるＬｅｆｔ ｒｉｇｈｔ ｄｅｔｅｒｍｉｎａｎｔ ｆａｃｔｏｒ１ （ＬＥＦＴＹ１）及びＬｅｆｔ ｒｉｇｈｔ ｄｅｔｅｒｍｉｎａｎｔ ｆａｃｔｏｒ２ （ＬＥＦＴＹ２）を見出すことに成功した。
<Left right dominant factor>
The proteomics method is a technique for developing proteins by two-dimensional electrophoresis and exhaustively searching for new biomarkers by mass spectrometry. However, the analysis has various limitations depending on the state of the frozen sample.
The inventors first obtained a formalin-fixed paraffin-embedded (FFPE) material of ovarian clear cell adenocarcinoma tissue derived from a patient with ovarian clear cell adenocarcinoma, and a section of a lesion containing clear cell adenocarcinoma cells by microdissection. Cut out. Next, a shotgun proteomic method was performed in which protein was extracted from the lesion, the protein was previously digested with trypsin, and directly measured and identified by mass spectrometry. By these series of methods, protein expression in the lesion can be efficiently analyzed, and the proteins left right factor 1 (LEFTY1) and left right factor 2 (LEFTY2) that are highly expressed in ovarian clear cell adenocarcinoma cells. I succeeded in finding it.
配列番号１で表されるアミノ酸配列は、ヒトのＬＥＦＴＹ１タンパク質のアミノ酸配列であり、配列番号２で表されるアミノ酸配列は、ヒトのＬＥＦＴＹ２タンパク質のアミノ酸配列である。
配列番号３で表される塩基配列は、ヒトのＬＥＦＴＹ１遺伝子の塩基配列（コーディング領域の配列）である。配列番号４で表される塩基配列は、ヒトのＬＥＦＴＹ２遺伝子の塩基配列（コーディング領域の配列）である。
ＬＥＦＴＹ１とＬＥＦＴＹ２のアミノ酸配列の相同性をMultain version 5.4.1(http://multalin.toulouse.inra.fr/multalin/multalin.html)によって求めたところ、約９６％であった。
The amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence of human LEFTY1 protein, and the amino acid sequence represented by SEQ ID NO: 2 is the amino acid sequence of human LEFTY2 protein.
The base sequence represented by SEQ ID NO: 3 is the base sequence of human LEFTY1 gene (coding region sequence). The base sequence represented by SEQ ID NO: 4 is the base sequence (coding region sequence) of the human LEFTY2 gene.
The homology between the amino acid sequences of LEFTY1 and LEFTY2 was determined by Multain version 5.4.1 (http://multalin.toulouse.inra.fr/multalin/multalin.html) to be about 96%.
≪卵巣明細胞腺癌細胞の検出方法≫
従って、本発明の卵巣明細胞腺癌細胞の検出方法は、生体試料中の卵巣明細胞腺癌細胞の検出方法であって、以下の（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は以下の（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を腫瘍マーカーとして用いる工程Ａを有するものである。
（ａ）配列番号１又は２で表されるアミノ酸配列、
（ｂ）配列番号１又は２で表されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換、又は付加されているアミノ酸配列、
（ｃ）配列番号１又は２で表されるアミノ酸配列に対して、８０％以上の同一性を有するアミノ酸配列
≪Method for detecting ovarian clear cell adenocarcinoma cells≫
Therefore, the method for detecting ovarian clear cell adenocarcinoma cells of the present invention is a method for detecting ovarian clear cell adenocarcinoma cells in a biological sample, and has any one of the following amino acid sequences (a) to (c): The method comprises a step A in which a protein or a protein having a partial sequence of any of the following amino acid sequences (a) to (c) is used as a tumor marker.
(A) the amino acid sequence represented by SEQ ID NO: 1 or 2,
(B) in the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence in which one to several amino acids are deleted, substituted, or added;
(C) an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 2
ここで、トランケート型タンパク質に代表される前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質に由来するポリペプチド断片、であっても腫瘍マーカーとして用いることが可能であるので、工程Ａは、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を検出する工程Ａ１を有するものであってもよい。この場合、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質は、（ａ）に対する特異的抗体と反応しうるタンパク質であることがより好ましい。
前記（ｂ）における、欠失、置換、又は付加されてもよいアミノ酸の数としては、１ 〜３０個が好ましく、１ 〜１５個がより好ましく、１ 〜１０個が特に好ましく、１ 〜 ５個が最も好ましい。
配列番号１又は２で表されるアミノ酸配列に対する同一性は、８５％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましい。
Here, even a polypeptide fragment derived from a protein having any one of the amino acid sequences (a) to (c) typified by a truncated protein can be used as a tumor marker. A may include a step A1 for detecting a protein having a partial sequence of any one of the amino acid sequences (a) to (c). In this case, the protein having a partial sequence of any one of the amino acid sequences (a) to (c) is more preferably a protein capable of reacting with a specific antibody against (a).
In the above (b), the number of amino acids that may be deleted, substituted or added is preferably 1 to 30, more preferably 1 to 15, particularly preferably 1 to 10, and 1 to 5. Is most preferred.
The identity to the amino acid sequence represented by SEQ ID NO: 1 or 2 is preferably 85% or more, more preferably 90% or more, and further preferably 95% or more.
尚、塩基配列の同一性は、例えば、ＮＣＢＩ（Ｎａｔｉｏｎａｌ Ｃｅｎｔｅｒ ｏｆ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ Ｉｎｆｏｒｍａｔｉｏｎ）から提供されているＢＬＡＳＴＮのプログラムを用いて解析を行うことにより算出される。アミノ酸配列の同一性は、同じくＮＣＢＩから提供されているＢＬＡＳＴＰのプログラムを用いて算出することが挙げられる。 In addition, the identity of the base sequence is calculated, for example, by performing analysis using a BLASTN program provided by NCBI (National Center of Biotechnology Information). The identity of amino acid sequences can be calculated using the BLASTP program also provided by NCBI.
これらの前記アミノ酸配列（ｂ）及びアミノ酸配列（ｃ）は、例えば、生体試料を提供する患者間又は対立遺伝子間に存在するＬＥＦＴＹ１遺伝子若しくはＬＥＦＴＹ２遺伝子の多型、変異等を有するアミノ酸配列に相当するものである。 These amino acid sequences (b) and (c) correspond to, for example, amino acid sequences having polymorphisms, mutations, etc. of the LEFTY1 gene or the LEFTY2 gene that exist between patients or alleles that provide biological samples. Is.
前記生体試料とは、卵巣明細胞腺癌細胞の検出対象者から採取した生体試料であることが好ましい。生体試料としては、組織、細胞、体液等の区分によらずいずれのものも用いることができる。卵巣明細胞腺癌細胞の病変部を検出対象とすることを意図する場合、生体試料は腹腔及び腹膜内の臓器由来のものであることが好ましく、卵巣由来のものであることがより好ましい。また、後述の実施例で示すように、ＬＥＦＴＹ１及びＬＥＦＴＹ２タンパク質は卵巣明細胞腺癌細胞外に分泌され得る。したがって、生体試料として、尿、血液、血清、リンパ液、組織液等の体液を用いることもまた好ましく、利便性の観点から血清を用いることが特に好ましい。血清を用いることにより、卵巣明細胞腺癌細胞をより手軽に効率よく検出することが可能である。 The biological sample is preferably a biological sample collected from a subject to detect ovarian clear cell adenocarcinoma cells. Any biological sample can be used regardless of the type of tissue, cell, body fluid or the like. When it is intended to detect a lesion of ovarian clear cell adenocarcinoma cells, the biological sample is preferably derived from an organ in the abdominal cavity and peritoneum, and more preferably derived from an ovary. In addition, as shown in Examples described later, LEFTY1 and LEFTY2 proteins can be secreted outside ovarian clear cell adenocarcinoma cells. Therefore, it is also preferable to use body fluids such as urine, blood, serum, lymph, and tissue fluid as the biological sample, and it is particularly preferable to use serum from the viewpoint of convenience. By using serum, ovarian clear cell adenocarcinoma cells can be detected more easily and efficiently.
前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を腫瘍マーカーとして用いる工程Ａとは、一例として該タンパク質を検出する工程であって、例えば、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を検出する工程Ａ１であってもよい。又は、該タンパク質を間接的に検出する工程であって、例えば、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を規定するポリヌクレオチドの発現を検出する工程Ａ２であってもよい。
当該タンパク質又は当該ポリヌクレオチドの発現量を測定することで、卵巣明細胞腺癌細胞を検出することができる。例えば、卵巣明細胞腺癌に罹患していない者由来の生体試料を用いた群、及び卵巣明細胞腺癌患者由来の生体試料を用いた群でのタンパク質又はポリヌクレオチドの発現量を指標にして、検出対象者から採取した生体試料でのタンパク質又はポリヌクレオチドの発現量の値により卵巣明細胞腺癌細胞の検出を行うことができる。後述の実施例で示すように、卵巣明細胞腺癌細胞ではＬＥＦＴＹ１及びＬＥＦＴＹ２タンパク質の発現量が上昇するので、卵巣明細胞腺癌に罹患していない者由来の生体試料を用いた群と比較して、ＬＥＦＴＹ１及びＬＥＦＴＹ２の発現量が統計的に有意に上昇していることをもって、卵巣明細胞腺癌細胞の検出を判別することができる。
また、卵巣明細胞腺癌患者由来の生体試料を用いた群でのタンパク質又はポリヌクレオチドの発現量を指標にして発現量の閾値を定め、卵巣明細胞腺癌への罹患を判別しても良い。
Step A using a protein having any one of the amino acid sequences (a) to (c) or a protein having a partial sequence of any one of the amino acid sequences (a) to (c) as a tumor marker is an example. Detecting the protein as, for example, a protein having any one of the amino acid sequences (a) to (c), or a partial sequence of any one of the amino acid sequences (a) to (c) Process A1 which detects the protein which has may be sufficient. Or a step of indirectly detecting the protein, for example, a protein having any one of the amino acid sequences (a) to (c), or any one of the amino acid sequences (a) to (c). Step A2 of detecting the expression of a polynucleotide defining a protein having a partial sequence of
By measuring the expression level of the protein or the polynucleotide, ovarian clear cell adenocarcinoma cells can be detected. For example, using the expression level of protein or polynucleotide in a group using a biological sample derived from a person not suffering from ovarian clear cell adenocarcinoma and a group using a biological sample derived from a patient with ovarian clear cell adenocarcinoma as an index The ovarian clear cell adenocarcinoma cells can be detected based on the expression level of the protein or polynucleotide in the biological sample collected from the detection subject. As shown in the examples below, the expression level of LEFTY1 and LEFTY2 protein is increased in ovarian clear cell adenocarcinoma cells, so compared with the group using biological samples derived from persons not suffering from ovarian clear cell adenocarcinoma. Thus, the detection of ovarian clear cell adenocarcinoma cells can be determined by the statistically significant increase in the expression levels of LEFTY1 and LEFTY2.
In addition, the expression level threshold may be determined using the expression level of protein or polynucleotide in the group using biological samples derived from patients with clear cell adenocarcinoma of the ovary as an index to determine the morbidity of ovarian clear cell adenocarcinoma. .
卵巣明細胞腺癌への罹患の判別にあたっては、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を腫瘍マーカーとして用いることに加えて、その他の腫瘍マーカーとの組み合わせにより卵巣明細胞腺癌への罹患を判別しても良い。また、卵巣明細胞腺癌の腫瘍マーカーとしてはＨＮＦ−１βを挙げることができる。これらを組み合わせることで、卵巣明細胞腺癌への罹患判別の信頼性をより向上させることができる。 In determining the morbidity of ovarian clear cell adenocarcinoma, the protein having any one of the amino acid sequences (a) to (c) above, or the partial sequence of any one of the amino acid sequences (a) to (c) above, In addition to using the protein as a tumor marker, the morbidity of ovarian clear cell adenocarcinoma may be determined by combination with other tumor markers. Moreover, HNF-1β can be mentioned as a tumor marker for ovarian clear cell adenocarcinoma. By combining these, it is possible to further improve the reliability of morbidity determination for ovarian clear cell adenocarcinoma.
（工程Ａ１）
前記工程Ａは、前記生体試料中の前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を検出する工程Ａ１を有するものであってもよい。
生体試料中から当該タンパク質を検出する方法としては特に制限されず、本分野の当業者において一般的な方法により実施することができる。一例として、ウェスタンブロット法、ＥＬＩＳＡ法、免疫染色等が挙げられる。また、多数の因子とともに当該タンパク質を検出する場合、抗体チップ等を用いてもよい。
(Process A1)
The step A comprises a protein having any one of the amino acid sequences (a) to (c) in the biological sample, or a protein having a partial sequence of any one of the amino acid sequences (a) to (c). You may have process A1 to detect.
The method for detecting the protein from the biological sample is not particularly limited, and can be carried out by a general method for those skilled in the art. Examples include Western blotting, ELISA, and immunostaining. In addition, when detecting the protein together with many factors, an antibody chip or the like may be used.
タンパク質の検出には、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質に対する特異的抗体を用いることが好ましい。
特異的抗体とは、ＬＥＦＴＹ１又はＬＥＦＴＹ２には結合するが、類似のタンパク質等の他の分子に対しては結合しない抗体か、又は、他の分子に結合する場合であっても卵巣明細胞腺癌細胞の検出が可能な程度の極めて低い程度でしか他のタンパク質に対しては結合しない抗体を意味する。
特異的抗体は、本分野の当業者において一般的な方法により得ることが可能である。例えば、（ａ）〜（ｃ）のいずれかのアミノ酸配列に基づいて化学合成されたポリペプチドを抗原として使用し、任意のヒト以外の動物へと免疫して目的の抗体を生産させ、動物の血清などに含まれる目的の特異的抗体のポリクローナル抗体を得ることができる。又は、目的の抗体を生産するハイブリドーマを作製するなどして、目的の特異的抗体のモノクローナル抗体を得てもよい。
抗原として使用するポリペプチドのアミノ酸配列は、（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列とすることができ、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を検出可能なように適宜配列を選択すればよい。
For protein detection, a specific antibody against a protein having any one of the amino acid sequences (a) to (c) or a protein having a partial sequence of any one of the amino acid sequences (a) to (c) is used. It is preferable to use it.
Specific antibody is an antibody that binds to LEFTY1 or LEFTY2 but does not bind to other molecules such as similar proteins, or ovarian clear cell adenocarcinoma even when bound to other molecules It means an antibody that binds to other proteins only to a very low extent that allows detection of cells.
Specific antibodies can be obtained by methods common to those skilled in the art. For example, using a polypeptide chemically synthesized based on any one of the amino acid sequences of (a) to (c) as an antigen, immunizing any non-human animal to produce the desired antibody, A polyclonal antibody of a specific antibody of interest contained in serum or the like can be obtained. Alternatively, a monoclonal antibody of the target specific antibody may be obtained by preparing a hybridoma that produces the target antibody.
The amino acid sequence of the polypeptide used as the antigen can be a partial sequence of any one of the amino acid sequences (a) to (c), and the protein having any one of the amino acid sequences (a) to (c) Alternatively, the sequence may be appropriately selected so that a protein having a partial sequence of any of the amino acid sequences of (a) to (c) can be detected.
抗原として使用するポリペプチドのアミノ酸配列は、例えば、配列番号１又は２で表されるアミノ酸配列中の１８aa〜３６６aaの領域を含むポリペプチドとすることができる。又は、配列番号１又は２で表されるアミノ酸配列中の１８aa〜３６６aaの領域の領域からなるポリペプチドとすることができる。 The amino acid sequence of the polypeptide used as the antigen can be, for example, a polypeptide containing a region of 18 aa to 366 aa in the amino acid sequence represented by SEQ ID NO: 1 or 2. Or it can be set as the polypeptide which consists of the area | region of the area | region of 18aa-366aa in the amino acid sequence represented by sequence number 1 or 2.
また、特異的抗体は、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を検出可能な範囲において、タグ、放射性物質、蛍光分子、酵素等の任意の物質を有した状態とすることができる。また、特異的抗体を一次抗体とし、蛍光分子や酵素等で標識された２次抗体を用いて前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を検出してもよい。 Further, the specific antibody can detect a protein having any one of the amino acid sequences (a) to (c) or a protein having a partial sequence of any one of the amino acid sequences (a) to (c). In the range, any substance such as a tag, a radioactive substance, a fluorescent molecule, and an enzyme can be included. In addition, a protein having any one of the amino acid sequences (a) to (c) described above using a secondary antibody labeled with a fluorescent molecule or an enzyme as a specific antibody, or the above (a) to (a A protein having a partial sequence of any amino acid sequence of c) may be detected.
（工程Ａ２）
前記工程Ａは、生体試料中の前記タンパク質を規定するポリヌクレオチドの発現を検出する工程Ａ２を有するものであってもよい。タンパク質を規定するポリヌクレオチドとは、タンパク質をコードするＤＮＡから転写されたｍＲＮＡ、それに由来するｃＤＮＡ、及びそれらのヌクレオチド断片を例示することができる。
生体試料中から前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を規定するポリヌクレオチドの発現を検出する方法としては、特に制限されず、本分野の当業者において一般的な方法により実施することができる。一例として、ＲＴ−ＰＣＲ、リアルタイムＰＣＲ、サザンブロット法、ノーザンブロット法等が挙げられる。また、多数の因子とともに当該ポリヌクレオチドを検出する場合、ＤＮＡチップ、ＲＮＡチップ等を用いてもよい。
(Process A2)
The step A may include a step A2 of detecting expression of a polynucleotide that defines the protein in a biological sample. Examples of the polynucleotide defining the protein include mRNA transcribed from DNA encoding the protein, cDNA derived therefrom, and nucleotide fragments thereof.
Expression of a polynucleotide defining a protein having any one of the amino acid sequences (a) to (c) or a protein having a partial sequence of any one of the amino acid sequences (a) to (c) from a biological sample The method for detecting is not particularly limited, and can be carried out by a general method for those skilled in the art. Examples include RT-PCR, real-time PCR, Southern blotting, Northern blotting, and the like. Moreover, when detecting the said polynucleotide with many factors, you may use a DNA chip, an RNA chip, etc.
前記工程Ａ２は、以下の（ｄ）〜（ｆ）のいずれかの塩基配列に基づいて作製したプローブ及び／又はプライマーを用いて、前記タンパク質を規定するポリヌクレオチドの発現を検出する工程であることが好ましい。
（ｄ）配列番号３又は４で表される塩基配列、
（ｅ）配列番号３又は４で表される塩基配列において、１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列、
（ｆ）配列番号１又は２で表される塩基配列に対して、８０％以上、好ましくは８５％以上、より好ましくは９０％以上、さらに好ましくは９５％、特に好ましくは９８％以上の同一性を有する塩基配列
The step A2 is a step of detecting the expression of a polynucleotide defining the protein using a probe and / or primer prepared based on any of the following base sequences (d) to (f): Is preferred.
(D) the base sequence represented by SEQ ID NO: 3 or 4,
(E) a nucleotide sequence in which one to several nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 3 or 4;
(F) 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95%, particularly preferably 98% or more identity to the base sequence represented by SEQ ID NO: 1 or 2 Nucleotide sequence having
ここで、前記（ｄ）〜（ｆ）のいずれかの塩基配列に基づいて作製された配列とは、該塩基配列由来とする配列（例えば、ｍＲＮＡ配列、ｃＤＮＡ配列）に基づいて作製された配列を含むものとする。前記１〜数個のヌクレオチドとしては、１ 〜３０個が好ましく、１ 〜１５個がより好ましく、１ 〜１０個が特に好ましく、１ 〜 ５個が特に好ましい。 Here, the sequence prepared based on any one of the base sequences (d) to (f) is a sequence prepared based on a sequence derived from the base sequence (for example, mRNA sequence, cDNA sequence). Shall be included. The 1 to several nucleotides are preferably 1 to 30, more preferably 1 to 15, particularly preferably 1 to 10, and particularly preferably 1 to 5.
前記（ｄ）〜（ｆ）のいずれかの塩基配列に基づく配列の一例としては、（ｄ）〜（ｆ）のいずれかの塩基配列の部分配列、前記（ｄ）〜（ｆ）のいずれかの塩基配列と相補的な配列、前記（ｄ）〜（ｆ）のいずれかの塩基配列と相補的な配列の部分配列からなる配列を挙げることができる。
また、当該プローブ及び／又はプライマーの配列は、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を規定するポリヌクレオチドの発現を検出し得る範囲において、これらの配列に加えて、タグ等の任意の配列をさらに有していてもよい。また、当該プローブ及び／又はプライマーは、前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質を規定するポリヌクレオチドの発現を検出可能な範囲において、放射性物質、蛍光分子等の任意の物質を有した状態とすることができる。
As an example of the sequence based on any one of the base sequences (d) to (f), any of the partial sequences of the base sequences (d) to (f) and any one of the above (d) to (f) And a sequence consisting of a partial sequence of a sequence complementary to the base sequence of any of the above (d) to (f).
Further, the probe and / or primer sequence has a protein having any one of the amino acid sequences (a) to (c) or a partial sequence of any one of the amino acid sequences (a) to (c). In addition to these sequences, an arbitrary sequence such as a tag may be further included within the range in which the expression of the polynucleotide defining the protein can be detected. The probe and / or primer is a protein having any one of the amino acid sequences (a) to (c) or a protein having a partial sequence of any one of the amino acid sequences (a) to (c). As long as the expression of the prescribed polynucleotide can be detected, it can be in a state having an arbitrary substance such as a radioactive substance or a fluorescent molecule.
より具体的には、配列番号５で表される塩基配列、及び／又は、配列番号６で表される塩基配列を有するプローブ及び／又はプライマーを用いて前記タンパク質を規定するポリヌクレオチドの発現を検出することができる。 More specifically, the expression of the polynucleotide defining the protein is detected using a probe and / or primer having the base sequence represented by SEQ ID NO: 5 and / or the base sequence represented by SEQ ID NO: 6. can do.
本発明の卵巣明細胞腺癌細胞の検出方法によれば、生体試料中の卵巣明細胞腺癌細胞の存在に関する情報を容易に得ることができるため、卵巣癌を早期に発見することができる。 According to the method for detecting ovarian clear cell adenocarcinoma cells of the present invention, it is possible to easily obtain information on the presence of ovarian clear cell adenocarcinoma cells in a biological sample, so that ovarian cancer can be detected early.
≪卵巣明細胞腺癌診断薬、卵巣明細胞腺癌診断用プライマー≫
本発明の卵巣明細胞腺癌診断薬は、以下の（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、又は以下の（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質に対する特異的抗体を含有する。
（ａ）配列番号１又は２で表されるアミノ酸配列、
（ｂ）配列番号１又は２で表されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換、又は付加されているアミノ酸配列、
（ｃ）配列番号１又は２で表されるアミノ酸配列に対して、８０％以上の同一性を有するアミノ酸配列
特異的抗体としては、前記≪卵巣明細胞腺癌細胞の検出方法≫の（工程Ａ１）において説明したものを同様のものを用いることができる。
卵巣明細胞腺癌診断薬を用いることにより、ＬＥＦＴＹ１又はＬＥＦＴＹ２タンパク質の発現を検出することができ、さらには卵巣明細胞腺癌の罹患を診断することができる。
<< Diagnosis of ovarian clear cell adenocarcinoma, primer for diagnosis of ovarian clear cell adenocarcinoma >>
The diagnostic reagent for ovarian clear cell adenocarcinoma of the present invention is a protein having any one of the following amino acid sequences (a) to (c), or a partial sequence of any one of the following amino acid sequences (a) to (c): Contains a specific antibody against a protein having
(A) the amino acid sequence represented by SEQ ID NO: 1 or 2,
(B) in the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence in which one to several amino acids are deleted, substituted, or added;
(C) Amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 2 Specific antibodies include (Step A1) of << Method for detecting ovarian clear cell adenocarcinoma cells >>. The same thing as what was demonstrated in) can be used.
By using an ovarian clear cell adenocarcinoma diagnostic agent, the expression of LEFTY1 or LEFTY2 protein can be detected, and further, morbidity of ovarian clear cell adenocarcinoma can be diagnosed.
本発明の卵巣明細胞腺癌診断用プライマーは、配列番号５で表される塩基配列を有するプライマー、及び配列番号６で表される塩基配列を有するプライマーを含む。
当該プライマーとしては、例えば、配列番号５又は配列番号６で表される塩基配列の５’側に対応する配列番号７又は配列番号８中の配列を有するプライマー、配列番号５又は配列番号６で表される塩基配列の５’側に任意の塩基配列を有するプライマーを挙げることができる。当該プライマーの長さの目安としては、１５〜３０ｍｅｒ程度とすることができる。
卵巣明細胞腺癌診断用プライマーを用いることにより、ＬＥＦＴＹ１又はＬＥＦＴＹ２のｍＲＮＡ由来の配列を増幅することができ、ＬＥＦＴＹ１又はＬＥＦＴＹ２のｍＲＮＡ発現を検出し、卵巣明細胞腺癌の罹患を診断することができる。
The primer for diagnosing ovarian clear cell adenocarcinoma of the present invention includes a primer having the base sequence represented by SEQ ID NO: 5 and a primer having the base sequence represented by SEQ ID NO: 6.
Examples of the primer include a primer having a sequence in SEQ ID NO: 7 or SEQ ID NO: 8 corresponding to the 5 ′ side of the base sequence represented by SEQ ID NO: 5 or SEQ ID NO: 6, represented by SEQ ID NO: 5 or SEQ ID NO: 6 A primer having an arbitrary base sequence on the 5 ′ side of the base sequence to be prepared can be mentioned. As a standard of the length of the primer, it can be about 15 to 30 mer.
By using a primer for diagnosis of ovarian clear cell adenocarcinoma, it is possible to amplify a sequence derived from mRNA of LEFTY1 or LEFTY2, detect mRNA expression of LEFTY1 or LEFTY2, and diagnose morbidity of ovarian clear cell adenocarcinoma it can.
≪卵巣明細胞腺癌診断用プローブ≫
本発明の卵巣明細胞腺癌診断用プローブは、以下の（ｇ）〜（ｉ）のいずれかを含む。
（ｇ）配列番号７又は８で表される塩基配列の少なくとも一部を含む配列、又は配列番号７又は８で表される塩基配列と相補的な塩基配列の少なくとも一部を含む配列、を含むヌクレオチド断片、
（ｈ）配列番号７又は８で表される塩基配列において１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列の少なくとも一部を含む配列、
又は配列番号７又は８で表される塩基配列において１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列と相補的な塩基配列の少なくとも一部を含む配列、を含むヌクレオチド断片、
（ｉ）配列番号７又は８で表される塩基配列の少なくとも一部を含む配列、又は配列番号７又は８で表される塩基配列と相補的な塩基配列の少なくとも一部を含む配列を有するポリヌクレオチドと、ストリンジェントな条件下でハイブリダイズするポリヌクレオチド又はヌクレオチド断片
≪Observed probe for clear cell adenocarcinoma≫
The probe for ovarian clear cell adenocarcinoma diagnosis of the present invention includes any of the following (g) to (i).
(G) including a sequence including at least a part of the base sequence represented by SEQ ID NO: 7 or 8, or a sequence including at least a part of the base sequence complementary to the base sequence represented by SEQ ID NO: 7 or 8. Nucleotide fragments,
(H) a sequence comprising at least a part of a base sequence in which 1 to several nucleotides are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 7 or 8;
Or a nucleotide fragment comprising a sequence comprising at least a part of a base sequence complementary to a base sequence in which one to several nucleotides are deleted, substituted or added in the base sequence represented by SEQ ID NO: 7 or 8 ,
(I) A poly having a sequence containing at least a part of the base sequence represented by SEQ ID NO: 7 or 8, or a sequence containing at least a part of the base sequence complementary to the base sequence represented by SEQ ID NO: 7 or 8 A polynucleotide or nucleotide fragment that hybridizes with a nucleotide under stringent conditions
配列番号７で表される塩基配列は、ヒトのＬＥＦＴＹ１遺伝子のｍＲＮＡに対応するＤＮＡ配列であり、配列番号８で表される塩基配列は、ヒトのＬＥＦＴＹ２遺伝子のｍＲＮＡに対応するＤＮＡ配列である。
配列番号７又は８で表される塩基配列と相補的な塩基配列を有するポリヌクレオチドは、例えば、それぞれＬＥＦＴＹ１遺伝子のｍＲＮＡ又はＬＥＦＴＹ２遺伝子のｍＲＮＡとハイブリダイズし、ＬＥＦＴＹ１遺伝子のｍＲＮＡ又はＬＥＦＴＹ２遺伝子のｍＲＮＡを検出することができる。したがって、相補的な塩基配列は配列番号７又は８で表される塩基配列の少なくとも一部を含む部分配列でもよく、部分配列の長さの目安としては、１５ｍｅｒ〜５０００ｍｅｒ程度、好ましくは２００ｍｅｒ〜１０００ｍｅｒ程度とすることができる。
配列番号７又は８で表される塩基配列を有するポリヌクレオチドは、例えば、それぞれＬＥＦＴＹ１遺伝子のｃＤＮＡ又はＬＥＦＴＹ２遺伝子のｃＤＮＡとハイブリダイズし得るので、ＬＥＦＴＹ１遺伝子のｃＤＮＡ又はＬＥＦＴＹ２遺伝子のｃＤＮＡを検出することができる。したがって、塩基配列は配列番号７又は８で表される塩基配列の少なくとも一部を含む部分配列でもよく、部分配列の長さの目安としては、１５ｍｅｒ〜５０００ｍｅｒ程度、好ましくは２００ｍｅｒ〜１０００ｍｅｒ程度、さらに好ましくは１５ｍｅｒ〜５００ｍｅｒ程度、特に好ましくは１５ｍｅｒ〜４０ｍｅｒ程度とすることができる。
The base sequence represented by SEQ ID NO: 7 is a DNA sequence corresponding to mRNA of human LEFTY1 gene, and the base sequence represented by SEQ ID NO: 8 is a DNA sequence corresponding to mRNA of human LEFTY2 gene.
The polynucleotide having a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 7 or 8, for example, hybridizes with the mRNA of LEFTY1 gene or the mRNA of LEFTY2 gene, respectively, and the mRNA of LEFTY1 gene or the mRNA of LEFTY2 gene is used. Can be detected. Therefore, the complementary base sequence may be a partial sequence including at least a part of the base sequence represented by SEQ ID NO: 7 or 8, and the length of the partial sequence is about 15 mer to 5000 mer, preferably 200 mer to 1000 mer. Can be about.
The polynucleotide having the base sequence represented by SEQ ID NO: 7 or 8 can hybridize with, for example, the LEFTY1 gene cDNA or the LEFTY2 gene cDNA, respectively, so that the LEFTY1 gene cDNA or the LEFTY2 gene cDNA can be detected. it can. Therefore, the base sequence may be a partial sequence including at least a part of the base sequence represented by SEQ ID NO: 7 or 8. As a measure of the length of the partial sequence, about 15 mer to 5000 mer, preferably about 200 mer to 1000 mer, Preferably, it is about 15 mer to 500 mer, particularly preferably about 15 mer to 40 mer.
前記（ｈ）において、欠失、置換、又は付加されてもよいヌクレオチドの数としては、１ 〜３０個が好ましく、１ 〜１５個がより好ましく、１ 〜１０個が特に好ましく、１ 〜 ５個が最も好ましい。
また、当該卵巣明細胞腺癌診断用プローブは、卵巣明細胞腺癌を診断可能な範囲において、放射性物質、蛍光分子等の任意の物質を有した状態とすることができる。
In the above (h), the number of nucleotides that may be deleted, substituted or added is preferably 1 to 30, more preferably 1 to 15, particularly preferably 1 to 10, and 1 to 5. Is most preferred.
Further, the ovarian clear cell adenocarcinoma diagnostic probe can be in a state having an arbitrary substance such as a radioactive substance or a fluorescent molecule within a range in which ovarian clear cell adenocarcinoma can be diagnosed.
前記（ｉ）におけるストリンジェントな条件下とは、例えば、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ−Ａ ＬＡＢＯＲＡＴＯＲＹ ＭＡＮＵＡＬ ＴＨＩＲＤ ＥＤＩＴＩＯＮ（Sambrookら、Cold Spring Harbor Laboratory Press）に記載の方法が挙げられる。例えば、５×ＳＳＣ（２０×ＳＳＣの組成：３Ｍ 塩化ナトリウム,０．３Ｍ クエン酸溶液,ｐＨ７．０）、０．１重量％ Ｎ−ラウロイルサルコシン、０．０２重量％のＳＤＳ、２重量％の核酸ハイブルダイゼーション用ブロッキング試薬、及び５０％フォルムアミドから成るハイブリダイゼーションバッファー中で、５５〜７０℃で数時間から一晩インキュベーションを行うことによりハイブリダイズさせる条件を挙げることができる。なお、インキュベーション後の洗浄の際に用いる洗浄バッファーとしては、好ましくは０．１重量％ＳＤＳ含有１×ＳＳＣ溶液、より好ましくは０．１重量％ＳＤＳ含有０．１×ＳＳＣ溶液である。 Examples of the stringent conditions in (i) above include the method described in Molecular Cloning-A Laboratory Manual THIRD EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). For example, 5 × SSC (composition of 20 × SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 0.1 wt% N-lauroyl sarcosine, 0.02 wt% SDS, 2 wt% The hybridization conditions can be exemplified by incubation for several hours to overnight at 55 to 70 ° C. in a hybridization buffer comprising 50% formamide and a blocking reagent for nucleic acid hybridization. The washing buffer used for washing after incubation is preferably a 0.1 × SSC solution containing 0.1 wt% SDS, more preferably a 0.1 × SSC solution containing 0.1 wt% SDS.
≪卵巣明細胞腺癌診断用キット≫
本発明の卵巣明細胞腺癌診断用キットは、本発明の卵巣明細胞腺癌診断用プライマー、本発明の卵巣明細胞腺癌診断用プローブ、又は前記（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質若しくは前記（ａ）〜（ｃ）のいずれかのアミノ酸配列の部分配列を有するタンパク質に対する特異的抗体、を含むものである。
卵巣明細胞腺癌診断用プライマーとしては、前記≪卵巣明細胞腺癌診断用プライマー≫で例示したものを同様のものを用いることができる。卵巣明細胞腺癌診断用プローブとしては、前記≪卵巣明細胞腺癌診断用プローブ≫で例示したものを同様のものを用いることができる。特異的抗体としては、前記≪卵巣明細胞腺癌細胞の検出方法≫の（工程Ａ１）において説明したものを同様のものを用いることができる。
また、卵巣明細胞腺癌診断をより簡便にするという観点から、本発明の卵巣明細胞腺癌診断用キットは、ＬＥＦＴＹ１又はＬＥＦＴＹ２タンパク質或いはＬＥＦＴＹ１又はＬＥＦＴＹ２ｍＲＮＡの検出に必要な試薬類、器具類等を備えていることが好ましい。ＬＥＦＴＹ１又はＬＥＦＴＹ２ｍＲＮＡの検出に必要な試薬類、器具類等は、ＬＥＦＴＹ１又はＬＥＦＴＹ２のｍＲＮＡに由来する配列の増幅に必要な試薬類、器具類等であってもよい。このように、卵巣明細胞腺癌細胞の検出に必要な試薬類をキット化することにより、卵巣明細胞腺癌診断をより簡便に行うことができる。
≪Ovary clear cell adenocarcinoma diagnostic kit≫
The kit for diagnosing ovarian clear cell adenocarcinoma of the present invention is the primer for diagnosing ovarian clear cell adenocarcinoma of the present invention, the probe for diagnosing ovarian clear cell adenocarcinoma of the present invention, or any one of the above (a) to (c) A specific antibody against a protein having an amino acid sequence or a protein having a partial sequence of any one of the amino acid sequences (a) to (c) above.
As the primer for diagnosing ovarian clear cell adenocarcinoma, the same primers as those exemplified in the above << Primer for ovarian clear cell adenocarcinoma diagnosis >> can be used. As the ovarian clear cell adenocarcinoma diagnostic probe, the same probes as those exemplified in the above-mentioned << Ovary clear cell adenocarcinoma diagnostic probe >> can be used. As the specific antibody, the same antibodies as those described in (Step A1) of << Method for detecting ovarian clear cell adenocarcinoma cells >> can be used.
Further, from the viewpoint of making ovarian clear cell adenocarcinoma diagnosis easier, the kit for diagnosing ovarian clear cell adenocarcinoma of the present invention comprises reagents, instruments and the like necessary for the detection of LEFTY1 or LEFTY2 protein or LEFTY1 or LEFTY2 mRNA. It is preferable to provide. Reagents, instruments, etc. necessary for detection of LEFTY1 or LEFTY2 mRNA may be reagents, instruments, etc. necessary for amplification of sequences derived from LEFTY1 or LEFTY2 mRNA. Thus, the diagnosis of ovarian clear cell adenocarcinoma can be performed more simply by preparing reagents necessary for the detection of ovarian clear cell adenocarcinoma cells.
次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
＜Shotgun proteomics＞
北里病院において２００６年以降に採取された卵巣癌の病理検体（ホルマリン固定パラフィン包埋（FFPE））のClear Cell（明細胞）、Endometrioid（類内膜性）、Serous（漿液性）、Mucinous（粘液性）の４種卵巣癌の検体から各４つ（合計１６サンプル）を選定した。次いで、レーザーマイクロダイセクションにより明細胞腺癌の検体から、明細胞腺癌細胞のみを狙って切り出した。他の各検体の切片からも同様に、病変部の癌細胞のみを切り出した。その後、各サンプルから切り出した病変部の癌細胞からタンパク質を抽出し、予めタンパク質をトリプシンで分解し、直接質量分析で測定・同定するショットガンプロテオミクス法を行った。
その結果、全４種の癌細胞の検体から約３０００種のタンパク質が得られ、そのうち、Clear Cellで高発現し、他のEndometrioid、Serous、Mucinousでは発現の低いタンパク質が、６６種あることが判明した。更に解析を進め、この明細胞腺癌細胞特異的に発現している６６種のタンパク質のうち、ＬＥＦＴＹ１及びＬＥＦＴＹ２が明細胞腺細胞において再現性よく発現していることを突き止めた。
<Shotgun proteomics>
Clear cell, clear cell, endometrioid, serous, mucinous (mucus) of pathological specimens of ovarian cancer (formalin fixed paraffin embedded (FFPE)) collected at Kitasato Hospital since 2006 4 types (total 16 samples) were selected from 4 types of ovarian cancer specimens. Subsequently, only clear cell adenocarcinoma cells were cut out from the clear cell adenocarcinoma specimen by laser microdissection. Similarly, only the cancer cells in the affected area were cut out from the sections of other specimens. Thereafter, a protein was extracted from the cancer cells of the lesioned part cut out from each sample, the protein was previously decomposed with trypsin, and a shotgun proteomics method was performed in which the protein was directly measured and identified by mass spectrometry.
As a result, about 3000 types of proteins were obtained from all 4 types of cancer cell specimens, of which 66 were found to be highly expressed in Clear Cell and 66 types of low expression in other Endometrioid, Serous and Mucinous. did. Further analysis was conducted, and it was found that among 66 proteins expressed specifically in clear cell adenocarcinoma cells, LEFTY1 and LEFTY2 were expressed with high reproducibility in clear cell gland cells.
＜免疫染色によるＬＥＦＴＹタンパク質の発現解析＞
Clear Cell、Endometrioid、Serous、Mucinousの各組織型の病理検体に対し、ＬＥＦＴＹタンパク質に対する免疫染色（IHC）を行った。解析には、ＬＥＦＴＹ１及びＬＥＦＴＹ２の両タンパク質に対して結合可能な抗体ab22569（Abcam社製）を用いた。
免疫染色の結果を図１に示す。卵巣明細胞腺癌において腫瘍細胞の細胞質から細胞膜にかけてＬＥＦＴＹ１/２タンパク質の強い染色性が認められたのに対し（上段、Clear Cell）、他の組織型では弱陽性もしくは陰性であった（下段、Endometrioid（類内膜性）、Serous（漿液性）、Mucinous（粘液性））。
<Expression analysis of LEFTY protein by immunostaining>
Immunostaining (IHC) for LEFTY protein was performed on pathological specimens of each tissue type of Clear Cell, Endometrioid, Serous, and Mucinous. For the analysis, an antibody ab22569 (manufactured by Abcam) capable of binding to both LEFTY1 and LEFTY2 proteins was used.
The result of immunostaining is shown in FIG. In ovarian clear cell adenocarcinoma, strong staining of LEFTY1 / 2 protein was observed from the cytoplasm of the tumor cells to the cell membrane (upper, Clear Cell), whereas other tissue types were weakly positive or negative (lower, Endometrioid (endometrioid), Serous (serous), Mucinous (mucous)).
上記の免疫染色の結果を定量的に算出した。染色強度（０： ｎｅｇａｔｉｖｅ、１： ｗｅａｋ、２： ｍｏｄｅｒａｔｅ、３： ｓｔｒｏｎｇ）と陽性範囲（１： １〜２５％、２： ２６〜５０％、３： ５１〜７５％、４： ７６〜１００％）を掛け合わせたスコア（ＩＨＣ ｓｃｏｒｅ ＝ ０ − １２）を算出した結果、明細胞腺癌は他の組織型に比して有意に高発現であることが明らかとなった（図２）。 The above immunostaining results were calculated quantitatively. Staining intensity (0: negative, 1: weak, 2: moderate, 3: strong) and positive range (1: 1-25%, 2: 26-50%, 3: 51-75%, 4: 76-100% ) Multiplied by (IHC score = 0-12), it was found that clear cell adenocarcinoma is significantly more expressed than other tissue types (Fig. 2).
＜Western blot法によるＬＥＦＴＹタンパク質の発現解析＞
ＬＥＦＴＹタンパク質と卵巣明細胞腺癌の特徴的分子であるHNF-1βとを、卵巣明細胞腺癌細胞株（ES2, OVTOKO, OVISE, KOC7C, TOV21G）を用いてWestern blot法により検出を行った。ＬＥＦＴＹタンパク質に対する抗体は、上述の免疫染色で用いたものと同じものを使用した。コントロールにはβ-actinを用いた。
Western Blotの結果を図３に示す。OVTOKO, OVISE, KOC7C, TOV21Gで、HNF-1βとＬＥＦＴＹ１/2タンパク質が検出された。これにより、ＬＥＦＴＹ１及びＬＥＦＴＹ２タンパク質が卵巣明細胞腺癌の腫瘍マーカーとして利用可能であることが示された。
<Expression analysis of LEFTY protein by Western blot method>
The LEFTY protein and HNF-1β, which is a characteristic molecule of ovarian clear cell adenocarcinoma, were detected by Western blotting using ovarian clear cell adenocarcinoma cell lines (ES2, OVTOKO, OVISE, KOC7C, TOV21G). The antibody against LEFTY protein was the same as that used in the above immunostaining. Β-actin was used as a control.
The results of Western Blot are shown in FIG. HNF-1β and LEFTY1 / 2 protein were detected in OVTOKO, OVISE, KOC7C, and TOV21G. Thus, it was shown that LEFTY1 and LEFTY2 proteins can be used as tumor markers for ovarian clear cell adenocarcinoma.
＜ＲＴ−ＰＣＲによるＬＥＦＴＹ ｍＲＮＡの発現解析＞
上記のWestern blot法による解析と同じ系を用いて、ＲＴ−ＰＣＲによりｍＲＮＡの検出を行った。ＬＥＦＴＹ１のｃＤＮＡの増幅に用いたプライマーの配列は、配列番号５（５’- ＴＧＣＴＡＣＡＧＧＴＧＴＣＧＧＴＧＣＡＧＡＧＧ -３’）及び配列番号６（５’- ＡＧＡＡＡＣＧＧＡＣＡＣＴＴＧＡＡＧＧＣＣＡＧＧ -３’）である。コントロールにはＧＡＰＤＨ遺伝子を用いた。
ＲＴ−ＰＣＲの結果を図４に示す。OVTOKO, OVISE, KOC7C, TOV21Gで、HNF-1βとＬＥＦＴＹ１のｍＲＮＡが検出された。これにより、ＬＥＦＴＹ１mRNAが卵巣明細胞腺癌の腫瘍マーカーとして利用可能であることが示された。
<Expression analysis of LEFTY mRNA by RT-PCR>
MRNA was detected by RT-PCR using the same system as the analysis by the Western blot method described above. The sequences of the primers used for amplification of the LEFTY1 cDNA are SEQ ID NO: 5 (5′-TGCTACAGGTGTCGGTGCAGAGGG-3 ′) and SEQ ID NO: 6 (5′-AGAACGCGACTACTGAAGGCCAGG-3 ′). The GAPDH gene was used as a control.
The results of RT-PCR are shown in FIG. HNF-1β and LEFTY1 mRNA were detected in OVTOKO, OVISE, KOC7C, and TOV21G. Thus, it was shown that LEFTY1 mRNA can be used as a tumor marker for ovarian clear cell adenocarcinoma.
＜ＬＥＦＴＹの遺伝子導入細胞の培養上清におけるＬＥＦＴＹタンパク質の検出＞
卵巣明細胞腺癌細胞株（OVISE, TOV21G）を24-well plateに1x105個まき、無血清培地にて48時間培養した後の培養上清（コントロールとして培地のみ）からタンパク質を抽出し、western blot法にてＬＥＦＴＹタンパク質の検出を行った。その結果、様々な長さのＬＥＦＴＹ1/2が検出され、卵巣癌細胞から細胞外へＬＥＦＴＹが分泌されることが明らかとなった（図５）。
<Detection of LEFTY protein in culture supernatant of LEFTY-transfected cells>
Ovarian clear cell adenocarcinoma cell lines (OVISE, TOV21G) were seeded on a 24-well plate, 1x10 5 cells, cultured in serum-free medium for 48 hours, and then protein was extracted from the culture supernatant (medium only as a control). The LEFTY protein was detected by blotting. As a result, various lengths of LEFTY1 / 2 were detected, and it was revealed that LEFTY was secreted extracellularly from ovarian cancer cells (FIG. 5).
以上で説明した各実施形態における各構成及びそれらの組み合わせ等は一例であり、本発明の趣旨を逸脱しない範囲で、構成の付加、省略、置換、およびその他の変更が可能である。また、本発明は各実施形態によって限定されることはなく、請求項（クレーム）の範囲によってのみ限定される。 The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.
本発明の卵巣明細胞腺癌細胞の検出方法よれば、生体試料中の卵巣明細胞腺癌細胞の検出を容易に行うことができるため、生体試料由来の卵巣明細胞腺癌細胞の検出方法として、卵巣明細胞腺癌細胞の検査に用いることができる。また、本発明の卵巣明細胞腺癌診断薬等は、卵巣明細胞腺癌の診断に用いる試薬類、検査機器として利用可能である。また、新薬及び新規治療法の開発にも応用することができる。 According to the method for detecting ovarian clear cell adenocarcinoma cells of the present invention, it is possible to easily detect ovarian clear cell adenocarcinoma cells in a biological sample. It can be used for the examination of ovarian clear cell adenocarcinoma cells. Further, the diagnostic agent for ovarian clear cell adenocarcinoma of the present invention can be used as reagents and test equipment used for diagnosis of ovarian clear cell adenocarcinoma. It can also be applied to the development of new drugs and new therapies.
Claims (12)
卵巣明細胞腺癌細胞の検出対象者から採取された卵巣組織の病理検体に対して、以下の（ａ）〜（ｃ）のいずれかのアミノ酸配列を有するタンパク質、以下の（ａ）〜（ｂ）のいずれかのアミノ酸配列の部分配列を有するタンパク質、又は、以下の（ｃ）のアミノ酸配列のうち配列番号１若しくは２で表されるアミノ酸配列に対して、同一性を有するアミノ酸配列の部分配列を有するタンパク質を腫瘍マーカーとして用いる工程Ａを有することを特徴とする卵巣明細胞腺癌細胞の検出方法。
（ａ）配列番号１又は２で表されるアミノ酸配列、
（ｂ）配列番号１又は２で表されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換、又は付加されているアミノ酸配列、
（ｃ）配列番号１又は２で表されるアミノ酸配列に対して、８０％以上の同一性を有するアミノ酸配列 A method for detecting ovarian clear cell adenocarcinoma cells in a biological sample, comprising:
A protein having any one of the following amino acid sequences (a) to (c) , and the following (a) to ( b ) for a pathological specimen of ovarian tissue collected from a detection subject of ovarian clear cell adenocarcinoma cells : ) Or a partial sequence of an amino acid sequence having identity to the amino acid sequence represented by SEQ ID NO: 1 or 2 among the amino acid sequences of (c) below: A method for detecting ovarian clear cell adenocarcinoma cells, comprising the step A of using a protein having the above as a tumor marker.
(A) the amino acid sequence represented by SEQ ID NO: 1 or 2,
(B) in the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence in which one to several amino acids are deleted, substituted, or added;
(C) an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 2
（ｄ）配列番号３又は４で表される塩基配列、
（ｅ）配列番号３又は４で表される塩基配列において、１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列、
（ｆ）配列番号３又は４で表される塩基配列に対して、８０％以上の同一性を有する塩基配列 The step A2 is a step of detecting expression of a polynucleotide defining the protein using a probe and / or a primer prepared based on any one of the following base sequences (d) to (f): Item 5. A method for detecting ovarian clear cell adenocarcinoma cells according to Item 4.
(D) the base sequence represented by SEQ ID NO: 3 or 4,
(E) a nucleotide sequence in which one to several nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 3 or 4;
(F) a base sequence having 80% or more identity to the base sequence represented by SEQ ID NO: 3 or 4
（ａ）配列番号１又は２で表されるアミノ酸配列、
（ｂ）配列番号１又は２で表されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換、又は付加されているアミノ酸配列、
（ｃ）配列番号１又は２で表されるアミノ酸配列に対して、８０％以上の同一性を有するアミノ酸配列 A protein having any one of the following amino acid sequences (a) to (c), which is used for a pathological specimen of ovarian tissue collected from a detection subject of ovarian clear cell adenocarcinoma cells, A protein having a partial sequence of any amino acid sequence of ( b ) or an amino acid sequence having identity to the amino acid sequence represented by SEQ ID NO: 1 or 2 of the following amino acid sequences of (c): A diagnostic agent for ovarian clear cell adenocarcinoma comprising a specific antibody against a protein having a partial sequence .
(A) the amino acid sequence represented by SEQ ID NO: 1 or 2,
(B) in the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence in which one to several amino acids are deleted, substituted, or added;
(C) an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 2
（ｇ）配列番号７又は８で表される塩基配列の少なくとも一部を含む塩基配列、又は配列番号７又は８で表される塩基配列と相補的な塩基配列の少なくとも一部を含む塩基配列、を含むヌクレオチド断片、
（ｈ）配列番号７又は８で表される塩基配列において１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列の少なくとも一部を含む塩基配列、又は配列番号７又は８で表される塩基配列において１〜数個のヌクレオチドが欠失、置換、又は付加されている塩基配列と相補的な塩基配列の少なくとも一部を含む塩基配列、を含むヌクレオチド断片、
（ｉ）配列番号７又は８で表される塩基配列の少なくとも一部を含む塩基配列、又は配列番号７又は８で表される塩基配列と相補的な塩基配列の少なくとも一部を含む塩基配列を有するポリヌクレオチドと、ストリンジェントな条件下でハイブリダイズするポリヌクレオチド又はヌクレオチド断片 Ovarian clear cell adenocarcinoma diagnosis characterized in that it is used for a pathological specimen of ovarian tissue collected from a detection subject of ovarian clear cell adenocarcinoma cells and includes any of the following (g) to (i): Probe.
(G) a base sequence comprising at least a part of the base sequence represented by SEQ ID NO: 7 or 8, or a base sequence comprising at least a part of the base sequence complementary to the base sequence represented by SEQ ID NO: 7 or 8. A nucleotide fragment comprising
(H) a nucleotide sequence comprising at least a part of a nucleotide sequence in which one to several nucleotides are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 7 or 8, or SEQ ID NO: 7 or 8 A nucleotide fragment comprising a base sequence comprising at least a part of a base sequence complementary to a base sequence in which 1 to several nucleotides are deleted, substituted or added in the represented base sequence,
(I) a base sequence comprising at least a part of the base sequence represented by SEQ ID NO: 7 or 8, or a base sequence comprising at least a part of the base sequence complementary to the base sequence represented by SEQ ID NO: 7 or 8 A polynucleotide or nucleotide fragment that hybridizes with a polynucleotide having a stringent condition
（ａ）配列番号１又は２で表されるアミノ酸配列、
（ｂ）配列番号１又は２で表されるアミノ酸配列において、１〜数個のアミノ酸が欠失、置換、又は付加されているアミノ酸配列、
（ｃ）配列番号１又は２で表されるアミノ酸配列に対して、８０％以上の同一性を有するアミノ酸配列 A protein having any one of the following amino acid sequences (a) to (c), which is used for a pathological specimen of ovarian tissue collected from a detection subject of ovarian clear cell adenocarcinoma cells, A protein having a partial sequence of any amino acid sequence of ( b ) or an amino acid sequence having identity to the amino acid sequence represented by SEQ ID NO: 1 or 2 of the following amino acid sequences of (c): A diagnostic kit for ovarian clear cell adenocarcinoma comprising a specific antibody against a protein having a partial sequence .
(A) the amino acid sequence represented by SEQ ID NO: 1 or 2,
(B) in the amino acid sequence represented by SEQ ID NO: 1 or 2, an amino acid sequence in which one to several amino acids are deleted, substituted, or added;
(C) an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 2
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