JP4677566B2 - Oncogene and diagnostic kit using the same - Google Patents
Oncogene and diagnostic kit using the same Download PDFInfo
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- JP4677566B2 JP4677566B2 JP2007500455A JP2007500455A JP4677566B2 JP 4677566 B2 JP4677566 B2 JP 4677566B2 JP 2007500455 A JP2007500455 A JP 2007500455A JP 2007500455 A JP2007500455 A JP 2007500455A JP 4677566 B2 JP4677566 B2 JP 4677566B2
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- C07K—PEPTIDES
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- C07K14/82—Translation products from oncogenes
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- C12Q2600/00—Oligonucleotides characterized by their use
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Description
本発明は、癌の分野に関する。具体的には癌遺伝子及びそれを利用した診断キットに関する。 The present invention relates to the field of cancer. Specifically, it relates to an oncogene and a diagnostic kit using the oncogene.
癌の代表的な例の一つである大腸癌（直腸癌および結腸癌）は、加齢と共に増加し、６０歳代をピークとする悪性腫瘍であって、リンパ行性、血行性、腹膜播種性などの転移を生じ、特に血行性転移によっては肝臓癌に進行することが多い。他の悪性腫瘍と同様、大腸癌に対する対策としては癌腫の早期発見が最も重要な課題である。特に大腸上部に発生した癌は自覚症状に乏しいため、発見時には既に病状が進行してしまっている危険性が高い。
従来、大腸癌に対しては主に便潜血反応によるスクリーニングと、ＣＥＡあるいはＣＡ１９−９等の血清マーカーによる診断、並びに治療経過の診断が行われている。
しかしながら、これらの方法はいずれも既に進行してしまっている癌（進行癌）では陽性率が高いものの、早期の段階の癌（早期癌）では陽性率が極めて低く、正確な診断が困難であった。
ここで、癌などの悪性腫瘍の簡便かつ確実な早期診断を可能とする方法として、癌組織特異的タンパク質マーカーを用いた分子生物学的診断方法が提案されている。この方法は大がかりな設備を必要とせず、被験体への負担も少ないため、自覚症状のない多くの被験体に対しても広範囲に実施することが可能である。例えば特開平７−５１０６５号公報には、糖タンパク質３９の腫瘍マーカーとしての利用が開示されている。
また一方で、大腸がんに関するＦＩＲ遺伝子、及びそれを用いた癌診断キットについての開示が国際公開第２００４／０１８６７９号公報に記載されている。
しかしながら、下記特許文献１に記載される糖タンパク質は、実際に腫瘍マーカーとして使用されることは記載されておらず、また、その精度において課題を残す。
また、上記特許文献２には大腸がんに関するＦＩＲ遺伝子及びその改変体についての示唆があり有用であるものの、その改変体の全貌の特定、腫瘍マーカーとしての精度について未だ改善の余地がある。早期発見が極めて重要である癌においては、腫瘍マーカーの機能として非常に高い精度が求められるのである。
即ち本発明は、以上のとおり事情に鑑み、大腸癌の診断により有効ながん遺伝子及びそれを用いた癌診断キットを提供することを目的とする。Colorectal cancer (rectal cancer and colon cancer), which is one of the typical examples of cancer, is a malignant tumor that increases with aging and peaks in the 60s, and is lymphatic, hematogenous, and peritoneal dissemination. Sexual metastasis occurs, and in particular, hematogenous metastasis often progresses to liver cancer. Like other malignant tumors, early detection of carcinoma is the most important issue as a countermeasure against colorectal cancer. In particular, cancer that has developed in the upper part of the large intestine has few subjective symptoms, so there is a high risk that the disease has already progressed when discovered.
Conventionally, screening for fecal occult blood, diagnosis with serum markers such as CEA or CA19-9, and diagnosis of the course of treatment have been performed for colorectal cancer.
However, although all of these methods have a high positive rate in cancer that has already progressed (advanced cancer), the positive rate is extremely low in early-stage cancer (early cancer), making accurate diagnosis difficult. It was.
Here, as a method that enables simple and reliable early diagnosis of malignant tumors such as cancer, a molecular biological diagnosis method using a cancer tissue-specific protein marker has been proposed. Since this method does not require large-scale equipment and places less burden on the subject, it can be performed over a wide range of subjects with no subjective symptoms. For example, JP-A-7-51065 discloses the use of glycoprotein 39 as a tumor marker.
On the other hand, the disclosure of the FIR gene relating to colorectal cancer and a cancer diagnostic kit using the same are described in International Publication No. 2004/018679.
However, it is not described that the glycoprotein described in Patent Document 1 below is actually used as a tumor marker, and a problem remains in its accuracy.
Moreover, although the above-mentioned Patent Document 2 is useful because it suggests the FIR gene related to colorectal cancer and its modified form, there is still room for improvement in the identification of the entire form of the modified form and the accuracy as a tumor marker. In cancer in which early detection is extremely important, a very high accuracy is required as a function of a tumor marker.
That is, in view of the circumstances as described above, an object of the present invention is to provide an oncogene that is effective in diagnosing colorectal cancer and a cancer diagnostic kit using the same.
本発明者らは、大腸癌患者の手術標本の正常部および癌部から本人の了解を得てタンパク質およびｍＲＮＡを抽出し、それらを解析することによって、大腸癌細胞において特異的に発現し、従来は腫瘍マーカーとして極めて高い精度で機能する２種の抗原ペプチドを見出し、本発明に想到した。即ち、具体的には以下の手段を採用する。
まず第一の手段として、下記（ａ）乃至（ｄ）のいずれかに記載のポリヌクレオチドとする。
（ａ）配列番号２に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、（ｂ）配列番号２に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列から１個又は複数個のアミノ酸が置換、付加、又は欠失したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、（ｃ）配列番号４に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド、（ｄ）配列番号４に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列から１個又は複数個のアミノ酸が置換、付加、又は欠失したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド
このようにすることで、癌を検出するためのマーカーとして用いることができる。
また第二の手段において、第一の手段におけるポリヌクレオチドを含んだ核酸分子とする。
また、第三の手段として、第一の手段におけるポリヌクレオチドと特異的な因子を用いる癌診断キットとする。なお、本明細書でいう「特異的な因子」とは、その生物学的因子（例えば、ポリヌクレオチド）に対する親和性が、他の無関連の（特に、同一性が３０％未満のもの。あるいは、ある特定の場合、同一性９９％未満のもの。さらに別の実施形態では、点変異のみの相違を有するものなど）生物学的因子（例えば、ポリヌクレオチドまたはポリペプチド）に対する親和性よりも有意に高いものをいう。そのような親和性は、例えば、ハイブリダイゼーションアッセイ、結合アッセイなどによって測定することができる。
またこの手段において、特異的な因子は、核酸分子、ポリペプチド、脂質、糖鎖、有機低分子、及びそれらの複合分子、の少なくともいずれかであること、癌診断キットが診断する癌は、直腸癌、結腸癌の少なくともいずれかであることが望ましい。
また、第四の手段として、採取した二つの試料それぞれに対し、下記（ａ）乃至（ｄ）のいずれかに記載のポリヌクレオチドの発現量を求めるステップ、その求めた発現量の比を算出するステップ、を有する方法とする。
（ａ）配列番号２に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド
（ｂ）配列番号２に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列から１個又は複数個のアミノ酸が置換、付加、又は欠失したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド
（ｃ）配列番号４に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列からなるタンパク質をコードするポリヌクレオチド
（ｄ）配列番号４に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠落したアミノ酸配列から１個又は複数個のアミノ酸が置換、付加、又は欠失したアミノ酸配列
なおこの場合において、試料の一方は非癌部組織から採取した試料とし、もう一方を例えば癌部組織であるとの疑いのある組織から採取した試料とし、この両者において発現量の比が異なる場合、癌について疑いのあるハイリスク者であると判定することができる。
またこの手段において、ポリヌクレオチドの発現を求めるステップは、ＰＣＲを用いてなること、発現量の比を算出するステップは、発現の有無を調べることであることも望ましい。
また第五の手段として、採取した二つの試料それぞれに対し、下記（ａ）乃至（ｄ）のいずれかに記載のアミノ酸配列からなるたんぱく質の発現量を求めるステップ、その求めた発現量の比を算出するステップ、を有する方法とする。
（ａ）配列番号２に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列
（ｂ）配列番号２に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列から１個又は複数個のアミノ酸が置換、付加、又は欠失したアミノ酸配列
（ｃ）配列番号４に記載のアミノ酸配列のうち第９位から第のアミノ酸３７位のアミノ酸が欠失したアミノ酸配列
（ｄ）配列番号４に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列から１個又は複数個のアミノ酸が置換、付加、又は欠失したアミノ酸配列
なおこの場合において、試料の一方は非癌部組織から採取した試料とし、もう一方を例えば癌部組織であるとの疑いのある組織から採取した試料とし、この両者において発現量の比が異なる場合、癌について疑いのあるハイリスク者であると判定することができる。
なおこの場合において、たんぱく質の発現を求めるステップは、ウエスタンブロットを用いてなること、採取した二つの試料のうち一つは正常な試料であること、更には、発現量の比を算出するステップは、発現の有無を調べることであることも望ましい。
以上により、きわめて高い精度で大腸癌の診断が可能ながん遺伝子及びそれを用いた癌診断キットを提供することができる。The present inventors obtained their consent from the normal part and cancer part of a surgical specimen of a colorectal cancer patient, extracted proteins and mRNA, analyzed them, and specifically expressed in colorectal cancer cells. Discovered two types of antigen peptides that function with extremely high accuracy as tumor markers, and arrived at the present invention. Specifically, the following means are employed.
First, as a first means, the polynucleotide described in any of (a) to (d) below is used.
(A) a polynucleotide encoding a protein comprising an amino acid sequence from which amino acids at positions 9 to 37 are deleted in the amino acid sequence set forth in SEQ ID NO: 2; (b) among the amino acid sequences set forth in SEQ ID NO: 2 A polynucleotide encoding a protein comprising an amino acid sequence in which one or more amino acids have been substituted, added, or deleted from the amino acid sequence from which amino acids at positions 9 to 37 have been deleted; (c) SEQ ID NO: 4 A polynucleotide encoding a protein comprising an amino acid sequence from which amino acids at positions 9 to 37 are deleted among the amino acid sequences described; (d) positions 9 to 37 in the amino acid sequence of SEQ ID NO: 4 A protein comprising an amino acid sequence in which one or more amino acids are substituted, added, or deleted from the amino acid sequence from Polynucleotide encoding By doing so, can be used as a marker for detecting cancer.
The second means is a nucleic acid molecule containing the polynucleotide in the first means.
Moreover, it is set as the cancer diagnostic kit which uses a polynucleotide and the specific factor in a 1st means as a 3rd means. As used herein, the term “specific factor” means that the affinity for the biological factor (for example, polynucleotide) is unrelated to other factors (particularly, those having an identity of less than 30%). , In certain cases, less than 99% identity, and in yet another embodiment, such as those having only point mutation differences) more significant than affinity for a biological agent (eg, polynucleotide or polypeptide). It is expensive. Such affinity can be measured, for example, by hybridization assays, binding assays, and the like.
In this means, the specific factor is at least one of a nucleic acid molecule, a polypeptide, a lipid, a sugar chain, a small organic molecule, and a complex molecule thereof. The cancer diagnosed by the cancer diagnostic kit is a rectum. Desirably, it is at least one of cancer and colon cancer.
As a fourth means, for each of the two collected samples, a step of obtaining the expression level of the polynucleotide according to any of the following (a) to (d), and a ratio of the obtained expression levels is calculated. Step.
(A) a polynucleotide encoding a protein comprising an amino acid sequence in which amino acids at positions 9 to 37 are deleted among the amino acid sequences set forth in SEQ ID NO: 2; and (b) the amino acid sequence set forth in SEQ ID NO: 2 A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids have been substituted, added, or deleted from the amino acid sequence in which the amino acids at positions 9 to 37 are deleted (c) described in SEQ ID NO: 4 Polynucleotide encoding a protein consisting of an amino acid sequence in which amino acids at positions 9 to 37 are deleted in the amino acid sequence (d) The amino acids at positions 9 to 37 in the amino acid sequence set forth in SEQ ID NO: 4 An amino acid sequence in which one or more amino acids are substituted, added, or deleted from the missing amino acid sequence. One sample is taken from a non-cancerous tissue, and the other sample is taken from a tissue suspected of being a cancerous tissue, for example. It can be determined that the person is at risk.
In this means, it is also desirable that the step of obtaining the expression of the polynucleotide is performed using PCR, and the step of calculating the expression level ratio is to examine the presence or absence of the expression.
Further, as a fifth means, for each of the two collected samples, a step of obtaining an expression level of a protein comprising the amino acid sequence described in any of (a) to (d) below, and a ratio of the obtained expression levels: A step of calculating.
(A) Amino acid sequence in which amino acids at positions 9 to 37 are deleted from the amino acid sequence described in SEQ ID NO: 2 (b) Amino acids at positions 9 to 37 in the amino acid sequence described in SEQ ID NO: 2 An amino acid sequence in which one or more amino acids have been substituted, added, or deleted from the amino acid sequence deleted from (c) the amino acid sequence from position 9 to position 37 in the amino acid sequence of SEQ ID NO: 4 Deleted amino acid sequence (d) One or more amino acids were substituted, added, or deleted from the amino acid sequence in which amino acids at positions 9 to 37 were deleted from the amino acid sequence shown in SEQ ID NO: 4. In this case, one of the samples is a sample collected from a non-cancerous tissue, and the other is a sample collected from a tissue suspected of being a cancerous tissue, for example. If the ratio of the current amount is different, it can be determined that the person is a high-risk person suspected of having cancer.
In this case, the step of obtaining the expression of the protein consists of using a Western blot, that one of the two collected samples is a normal sample, and further, the step of calculating the ratio of the expression levels is It is also desirable to examine the presence or absence of expression.
As described above, an oncogene capable of diagnosing colorectal cancer with extremely high accuracy and a cancer diagnostic kit using the same can be provided.
図１は、ＦＩＲ５４２、ＰＵＦ６０及びそれらの改変体についてのアミノ酸配列構造を示す図である。
図２は、実施例１におけるＲＴ−ＰＣＲの結果を示す図である。
図３は、図２に示す結果をより明確に示した図である。FIG. 1 is a diagram showing amino acid sequence structures of FIR542, PUF60, and their variants.
FIG. 2 shows the results of RT-PCR in Example 1.
FIG. 3 is a diagram showing the results shown in FIG. 2 more clearly.
以下、本発明の実施の形態について詳細に説明する。
（ＦＩＲの背景説明）
本明細書において「ＦＩＲ」とは、ＦＢＰ相互作用リプレッサーのことをいい、具体的には配列番号１又は配列番号２で特定される因子をいう。配列番号１はＦＩＲの塩基配列を示し、配列番号２はＦＩＲのアミノ酸配列を示す。配列番号１および２で特定されるＦＩＲは、本明細書においてＦＩＲ５４２とも称され、正常型の配列である。ＦＩＲの機能は、ｃ−ｍｙｃの発現抑制と報告されている。
ｃ−Ｍｙｃは、細胞の増殖、分化およびアポトーシスを調節する転写因子である。ｃ−Ｍｙｃ発現のアップレギュレーションおよびダウンレギュレーションの両方が、特定の状況下でアポトーシスを誘導し得る。本発明者らは、ｃ−ｍｙｃ遺伝子のリプレッサーであるＦＩＲ（ＦＢＰ相互作用リプレッサー）がＨｅＬａ細胞においてアポトーシスを誘導したことを確認した。ＦＩＲのアミノ末端抑制ドメインの欠失によってＦＩＲ駆動性のアポトーシスが起こらなくなり、このことは、細胞死がＦＩＲの転写標的（例えば、ｃ−ｍｙｃ）によって媒介されることを示唆する。結腸直腸腫瘍の評価によって、ＦＩＲ ｍＲＮＡレベルおよびＦＩＲタンパク質レベルの増加に関わらずｃ−ｍｙｃ発現の上昇（結腸直腸癌においてしばしば観察されることであるが）が明らかになった。ＦＩＲ媒介ｃ−ｍｙｃ抑制の排除およびＦＩＲ駆動性アポトーシスに対する耐性は、おそらく、最後に悪性結腸直腸癌となる多段階発癌の重要な工程を表す。
（ＦＩＲの改変体）
本実施形態は、ＦＩＲの改変体の一つに関する。従来知られていたＦＩＲの改変体の一つについての塩基配列を配列番号３に、そのアミノ酸配列を配列番号４に示す。
配列番号３および４で特定されるＦＩＲは、５５９アミノ酸配列の長さがあることからＦＩＲ５５９またはＰＵＦ６０と呼ばれる（本明細書では以下統一して「ＰＵＦ６０」ということとする。）。ＰＵＦ６０はＲＮＡスプライシングに関与しているとされており、配列番号２で示すＦＩＲ５４２のアミノ酸配列においては、第９８位のアミノ酸と第９９位のアミノ酸との間に１７アミノ酸に対応する塩基配列が挿入されている。
また、本明細書にて言及するＦＩＲの改変態について、そのアミノ酸配列の構成を図１に示す。
図１は、上記ＦＩＲ及びＰＵＦ６０、更にはその改変体を示すものであって、本明細書で言及するＦＩＲの改変体は、５３０のアミノ酸配列の長さを有しており、ＰＵＦ６０の塩基配列（配列番号４）の第９位から第３７位のアミノ酸が欠失した配列となっている（以下本明細書では「ＳｈｏｒｔＰＵＦ６０」と呼ぶ。図１（ｃ）参照）。
図１（ｄ）に示すＦＩＲの改変体は、５１３のアミノ酸配列の長さを有しており、ＳｈｏｒｔＰＵＦ６０がＰＵＦ６０に比べて欠失しているアミノ酸だけでなく、ＦＩＲがＰＵＦ６０に比べて欠失しているアミノ酸も欠失している構成となっている。即ち、ＰＵＦ６０のアミノ酸配列に対し、第９位〜第３７位（塩基配列では第２５位〜１１１位の塩基）、第９９位から第１１５位（塩基配列では第２９５位〜第３４５位の塩基）のアミノ酸が欠失した配列となっている（以下本明細書では「ＳｈｏｒｔＦＩＲ」と呼ぶ。図１（ｄ）参照）
これらの４つの改変体に加え、そのアミノ酸配列に置換が見られる改変体も見出された。ただしこの置換はＮ末端側の４００塩基の部位に存在する約１３５アミノ酸の領域に多く見られた。本明細書において使用されるポリヌクレオチドは、これによって発現されるポリペプチドが改変の基礎となるポリペプチドと実質的に同一の活性を有する限り、その配列の一部が欠失または他の塩基により置換されること、あるいは他の核酸配列が一部挿入されることも許容できる。
これらの改変体は、大腸癌組織と正常粘膜とを比較すると、いずれも発現が増大していることが見出され、癌の指標として使用することができると考えられるが、特に、本明細書にて言及するＳｈｏｒｔＰＵＦ６０、ＳｈｏｒｔＦＩＲについては極めて高い精度で指標として用いることができる。より具体的には、ＳｈｏｒｔＰＵＦ６０、ＳｈｏｒｔＦＩＲの二つについては、これら改変体の存在の有無のみを癌の指標とすることができる。
以下、実施例を示して本願の発明についてさらに詳細かつ具体的に説明する。しかし本発明の範囲は実施例のみに限定されるものではない。Hereinafter, embodiments of the present invention will be described in detail.
(FIR background explanation)
As used herein, “FIR” refers to an FBP interaction repressor, and specifically refers to a factor specified by SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1 shows the base sequence of FIR, and SEQ ID NO: 2 shows the amino acid sequence of FIR. The FIR identified by SEQ ID NOs: 1 and 2 is also referred to herein as FIR542 and is a normal sequence. The function of FIR has been reported to suppress c-myc expression.
c-Myc is a transcription factor that regulates cell proliferation, differentiation and apoptosis. Both up-regulation and down-regulation of c-Myc expression can induce apoptosis under certain circumstances. The present inventors confirmed that FIR (FBP interaction repressor), which is a repressor of c-myc gene, induced apoptosis in HeLa cells. Deletion of the amino terminal repression domain of FIR prevents FIR-driven apoptosis, suggesting that cell death is mediated by FIR transcriptional targets (eg, c-myc). Evaluation of colorectal tumors revealed an increase in c-myc expression (although often observed in colorectal cancer) despite increased FIR mRNA and FIR protein levels. Elimination of FIR-mediated c-myc suppression and resistance to FIR-driven apoptosis probably represents an important step in multistage carcinogenesis that ultimately leads to malignant colorectal cancer.
(Modified FIR)
This embodiment relates to one variant of FIR. The base sequence of one of the conventionally known FIR variants is shown in SEQ ID NO: 3, and the amino acid sequence thereof is shown in SEQ ID NO: 4.
The FIR identified by SEQ ID NOs: 3 and 4 is called FIR559 or PUF60 because it has a length of 559 amino acid sequences (hereinafter, collectively referred to as “PUF60” in this specification). PUF60 is said to be involved in RNA splicing, and in the amino acid sequence of FIR542 shown in SEQ ID NO: 2, a base sequence corresponding to 17 amino acids is inserted between the amino acid at position 98 and the amino acid at position 99 Has been.
Moreover, the structure of the amino acid sequence is shown in FIG. 1 about the modified state of FIR referred in this specification.
FIG. 1 shows the above-mentioned FIR and PUF60, and further variants thereof. The variant of FIR referred to in this specification has a length of 530 amino acid sequences, and the nucleotide sequence of PUF60. This is a sequence in which the amino acids at positions 9 to 37 of (SEQ ID NO: 4) are deleted (hereinafter referred to as “ShortPUF60” in the present specification, see FIG. 1C).
The variant of FIR shown in FIG. 1 (d) has a length of 513 amino acid sequences, and not only the amino acid in which ShortPUF60 is deleted compared to PUF60, but also the FIR is deleted compared to PUF60. The amino acid is also deleted. That is, with respect to the amino acid sequence of PUF60, positions 9 to 37 (base positions 25 to 111 in the base sequence), positions 99 to 115 (base positions 295 to 345 in the base sequence) ) Is deleted (hereinafter referred to as “ShortFIR” in this specification, see FIG. 1 (d)).
In addition to these four variants, a variant in which substitution was found in the amino acid sequence was also found. However, this substitution was frequently observed in a region of about 135 amino acids present at a 400 base site on the N-terminal side. As used herein, a polynucleotide may be partially deleted or other bases as long as the polypeptide expressed thereby has substantially the same activity as the polypeptide on which it is modified. It is permissible to be substituted, or to partially insert other nucleic acid sequences.
These variants are found to have increased expression when compared with colon cancer tissue and normal mucosa, and can be used as an indicator of cancer. The Short PUF 60 and the Short FIR referred to in the above can be used as indices with extremely high accuracy. More specifically, for two of ShortPUF60 and ShortFIR, only the presence or absence of these variants can be used as an indicator of cancer.
Hereinafter, the present invention will be described in more detail and specifically with reference to examples. However, the scope of the present invention is not limited to the examples.
本実施例では、ＦＩＲ及びＳｈｏｒｔＦＩＲをコードする遺伝子の転写産物（ｍＲＮＡ）の癌組織および正常組織での転写をＲＴ−ＰＣＲによって分析した。
（１）材料と方法
１０名の患者の了解のもと、手術により摘出した直後の癌部組織及び正常部組織から標本を採取し、−８０℃で凍結させて保存した（なお以下凍結して保存した標本を「凍結標本」という。）。そしてこの採取した凍結標本から別途適量取り出し、ＲＮｅａｓｙ Ｍｉｎｉ Ｋｉｔ（Ｑｉａｇｅｎ社製）を用いてｍＲＮＡを抽出した。
このｍＲＮＡ（５μｇ／２０μｌ）を、１ｓｔ Ｓｔｒａｎｄ ｃＤＮＡ Ｓｙｎｔｈｅｓｉｓ Ｋｉｔ ｆｏｒ ＲＴ−ＰＣＲ（ＡＭＶ、ｃａｔ ｎｏ．１ ４８３ １８８、Ｒｏｃｈｅ社製）によりｃＤＮＡに変換し、これをＰＣＲ法で増幅した。ＰＣＲ法の具体的条件は、順方向プライマーとして５’−ｇｇｃｃｃｃａｔｃａａｇａｇｃａｔｃ−３’（配列番号７）を、逆方向プライマーとして５’−ｇｇｇｇｃｔｇｇｇｃｃａｇｇｇｔｃａｇ−３’（配列番号８）を用いた。また温度は、９５℃で１分間処理した後、９４℃で１分間、５４℃で１分間、７２℃で１０分間の処理を３０回繰返し、更に、７２℃で７分間処理することによって行った。
（２）結果
図２にこの結果を示す。大腸癌患者の癌部組織（Ｔ）および非癌部組織（Ｎ）からのＦＩＲ ｍＲＮＡの転写を比較したところ、癌組織細胞においてＦＩＲ ｍＲＮＡ及びＳｈｏｒｔＦＩＲ ｍＲＮＡの特異的発現が確認された。本図においては、ＦＩＲの場合は正常部組織の場合で発現が少量見られたときであっても、ＳｈｏｒｔＦＩＲの場合では、正常部組織での発現は殆ど見られなかった。この結果から、ＦＩＲのｍＲＮＡ発現量を調べることに比べ、ＳｈｏｒｔＦＩＲのｍＲＮＡ発現量を調べることが検査方法としてより精度が高く、極めて優れていることを示していた。この結果は、ＦＩＲ及びその改変体のアミノ酸配列において第９位から第３７位のアミノ酸の欠失がより癌部において重要な因子として機能することを示唆していると思われる。In this example, transcription of a transcription product (mRNA) of a gene encoding FIR and ShortFIR in cancer tissue and normal tissue was analyzed by RT-PCR.
(1) Materials and methods Under the consent of 10 patients, specimens were collected from cancer tissue and normal tissue immediately after surgery and stored frozen at -80 ° C (hereinafter frozen). The preserved specimen is called “frozen specimen”.) Then, an appropriate amount was taken out from the collected frozen specimen, and mRNA was extracted using RNeasy Mini Kit (manufactured by Qiagen).
This mRNA (5 μg / 20 μl) was converted into cDNA by 1st Strand cDNA Synthesis Kit for RT-PCR (AMV, cat no. 1 483 188, manufactured by Roche) and amplified by the PCR method. The specific conditions of the PCR method were 5′-ggccccatagaagcatc-3 ′ (SEQ ID NO: 7) as a forward primer and 5′-ggggctggggcgggtcag-3 ′ (SEQ ID NO: 8) as a reverse primer. In addition, after the treatment at 95 ° C. for 1 minute, the treatment was carried out by repeating the treatment at 94 ° C. for 1 minute, 54 ° C. for 1 minute, 72 ° C. for 10 minutes 30 times, and further at 72 ° C. for 7 minutes. .
(2) Results FIG. 2 shows the results. When transcription of FIR mRNA from cancerous tissue (T) and non-cancerous tissue (N) of colorectal cancer patients was compared, specific expression of FIR mRNA and ShortFIR mRNA was confirmed in cancer tissue cells. In this figure, even in the case of FIR, even when a small amount of expression was observed in the normal tissue, in the case of Short FIR, almost no expression was observed in the normal tissue. From this result, it was shown that examining the mRNA expression level of ShortFIR is higher in accuracy and extremely superior as a test method compared with examining the FIR mRNA expression level. This result seems to suggest that the deletion of amino acids from the 9th position to the 37th position in the amino acid sequence of FIR and its variants functions as a more important factor in the cancerous part.
まず、抗原ペプチドＦＩＲ（配列番号１）及びＳｈｏｒｔＦＩＲの癌組織および正常組織での発現をウェスタンブロットによって分析した。
（１） 材料及び方法
（１−１）抗ＦＩＲ抗体の作成
事前にＦＩＲペプチドのアミノ酸配列（配列番号２）から抗原性、疎水性、反応性、ペプチドの特異性をＢＬＡＳＴサーチし、特異的抗原性ペプチドとして以下の２つのペプチドをＦｍｏｃ／ｔ−Ｂｏｃによる固相法で合成し、純度を保証するためＨＰＬＣにより分析し、質量分析装置を用いて分子量を同定した。
ＮＨ２−ＣＤＫＷＫＰＰＱＧＴＤＳＩＫＭＥ−ＣＮＨ２
ＮＨ２−ＣＥＶＹＤＱＥＲＦＤＮＳＤＬＳＡ−ＣＯＯＨ
この合成ペプチドを用いて、無菌帝王切開を受けたニュージーランド産Ｓ．Ｐ．Ｆ基準（無病原菌）のウサギを常法により免疫し、血清を抽出し、アファニティー精製した抗ＦＩＲ抗体を作成した。
（１−２）ウェスタンブロット分析
１０名の患者の了解のもと、手術により摘出した直後の癌部組織及び正常部組織から標本を採取し、−８０℃で凍結させて保存した（なお以下凍結して保存した標本を「凍結標本」という。）。
そして、採取した標本それぞれについて、凍結された状態から適量取り出し、９．５Ｍ Ｕｅｒａ、２％ ＣＨＡＰＳ、１％ ＤＴＴ溶液中でホモジェナイズした後、卓上高速遠心器（ベックマン社製）で１５０００ｇにて６０分間遠心し、上清（タンパク質溶液）を抽出し、吸光度によりタンパク質濃度を同定した。
そして癌部組織および正常部組織から得られた５０μｇを、８％ Ｔｒｉｓ／Ｇｌｙｃｉｎｅ ＳＤＳ−Ｐｏｌｙａｃｒｙｌａｍｉｄｅ ｇｅｌにて電気泳動させた。そして電気泳動したタンパク質をニトロセルロース膜にトランスファーし、上記（１−１）で作成した抗体を用いてウェスタンブロット解析を行った。
（２）結果
大腸癌患者の癌部組織（Ｔ）および正常部組織（Ｎ）を比較したところ、いずれの患者においても、癌部組織においてＦＩＲタンパク質の特異的発現が確認され、正常部組織（Ｎ）については特異的発現が殆ど確認されなかった。特に、ＳｈｏｒｔＦＩＲの場合は、ＦＩＲでは正常部組織でも発現が少量確認されてしまっている場合であっても正常部組織での発現は殆ど見られなかった。即ちこれは、ＦＩＲの発現量を調べることに対して、ＳｈｏｒｔＦＩＲの発現量を調べる方が検査方法としてより精度が高く、極めて優れていることを示す。
以上、上記実施例により、人間から組織を採取し、ＦＩＲ改変体の存在を分析することで、大腸癌を確実に発見することができ、そのための診断キットを提供することができる。First, the expression of antigen peptide FIR (SEQ ID NO: 1) and Short FIR in cancer tissue and normal tissue was analyzed by Western blot.
(1) Material and method (1-1) Preparation of anti-FIR antibody A BLAST search for the antigenicity, hydrophobicity, reactivity, and peptide specificity from the amino acid sequence of the FIR peptide (SEQ ID NO: 2) in advance and specific antigen The following two peptides were synthesized as sex peptides by a solid phase method using Fmoc / t-Boc, analyzed by HPLC to ensure purity, and molecular weight was identified using a mass spectrometer.
NH 2 -CDKWKPPQGTDSIKME-CNH 2
NH 2 -CEVYDQERFDDNDLSA-COOH
Using this synthetic peptide, New Zealand S. cerevisiae undergoing sterile caesarean section. P. F-standard (pathogenic bacteria) rabbits were immunized by a conventional method, serum was extracted, and an affinity purified anti-FIR antibody was prepared.
(1-2) Western blot analysis Under the consent of 10 patients, specimens were collected from cancer tissue and normal tissue immediately after excision by surgery, frozen at -80 ° C and stored (hereinafter frozen). Specimens stored in this way are called “frozen specimens”.)
For each sample collected, an appropriate amount was taken out from the frozen state, homogenized in 9.5M Uera, 2% CHAPS, 1% DTT solution, and then for 60 minutes at 15000 g with a tabletop high speed centrifuge (manufactured by Beckman). After centrifugation, the supernatant (protein solution) was extracted, and the protein concentration was identified by absorbance.
Then, 50 μg obtained from the cancer tissue and normal tissue was electrophoresed with 8% Tris / Glycine SDS-Polyacrylamide gel. The electrophoresed protein was transferred to a nitrocellulose membrane, and Western blot analysis was performed using the antibody prepared in (1-1) above.
(2) Results When the cancerous tissue (T) and normal tissue (N) of colorectal cancer patients were compared, specific expression of FIR protein was confirmed in the cancerous tissue in any patient, and normal tissue ( N) almost no specific expression was confirmed. In particular, in the case of Short FIR, almost no expression was observed in the normal tissue even when a small amount of expression was confirmed in the normal tissue by FIR. In other words, this indicates that the method of examining the expression level of ShortFIR is more accurate and extremely superior as a test method than the method of examining the expression level of FIR.
As described above, according to the above examples, colon cancer can be reliably detected by collecting tissues from humans and analyzing the presence of FIR variants, and a diagnostic kit for that purpose can be provided.
以上のとおり本発明は、より確実に大腸癌の診断を行うための遺伝子を提供し、さらにこれを用いてより正確な癌の診断を行うことができる診断キットを提供できる。
［配列表］
As described above, the present invention provides a gene for more reliably diagnosing colorectal cancer, and further provides a diagnostic kit that can be used to more accurately diagnose cancer.
[Sequence Listing]
Claims (1)
（ａ）配列番号２に記載のアミノ酸配列からなるタンパク質
（ｂ）配列番号２に記載のアミノ酸配列のうち第９位から第３７位のアミノ酸が欠失したアミノ酸配列からなるタンパク質A method for detecting colorectal cancer cells , wherein one of the two collected samples is a sample collected from a non-cancerous tissue, and the other sample is a sample to be detected. For the expression of the two proteins, antibodies against the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 were used, and the presence or absence of their expression was examined by Western blotting. The following two proteins were samples to be detected. A method for detecting colorectal cancer cells , characterized in that if the expression of the protein (b) below is not observed in a sample derived from a non-cancerous tissue, both are expressed and the colorectal cancer cells are determined.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 (b) a protein comprising an amino acid sequence in which the amino acids at positions 9 to 37 are deleted from the amino acid sequence set forth in SEQ ID NO: 2
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