JP4520951B2 - Insulin secretion inducer and insulin secretion induction composition - Google Patents
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Description
本発明は、糖尿病をはじめとする種々の代謝性疾患の治療に有効なインスリン分泌誘導剤及びインスリン分泌誘導組成物に関する。 The present invention relates to a variety of active insulin secretion inducer and insulin secretion-inducing composition for the treatment of metabolic diseases including diabetes.
今日本人のライフスタイルの欧米化により、糖尿病、高脂血症、高血圧や肥満といった生活習慣病がクローズアップされている。このような中で消化管は食事中の栄養分を吸収するだけでなく消化管ホルモンを産生する内分泌器官として注目されており、胃で産生されるグレリンの他、消化管から分泌されるグルカゴン様ペプチド-1(Glucagon-like peptide-1：GLP-1)や胃酸分泌抑制ポリペプチド(Gastric inhibitory polypeptide：GIP)などが既にクローニングされている。グレリンは摂食亢進作用を持ちエネルギー代謝と関連がある。GLP-1やGIPはインクレチンとして食事負荷により膵臓に働きインスリン分泌を誘導する。また、GIPは脂肪にも発現しておりGIPレセプターのノックアウトマウスは痩せる事からも肥満との関連も示唆されている。このように、消化管ホルモンはエネルギー代謝や摂食行動などに関わっており、生活習慣病においてその成因の新しいメカニズムの解明につながってきている。しかしながら、消化管ホルモンの全容は未だ明らかにされておらず、不明な部分も多い。 Due to the westernization of Japanese lifestyles, lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension and obesity are now being highlighted. Under such circumstances, the digestive tract is attracting attention as an endocrine organ that not only absorbs nutrients in the diet but also produces gastrointestinal hormones. In addition to ghrelin produced in the stomach, a glucagon-like peptide secreted from the digestive tract -1 (Glucagon-like peptide-1: GLP-1) and gastric acid secretion inhibitory polypeptide (GIP) have already been cloned. Ghrelin has a feeding-enhancing effect and is associated with energy metabolism. GLP-1 and GIP act as incretin on the pancreas by dietary load and induce insulin secretion. In addition, GIP is also expressed in fat and GIP receptor knockout mice are thin, suggesting an association with obesity. Thus, gastrointestinal hormones are involved in energy metabolism and feeding behavior, and have led to the elucidation of new mechanisms of their origin in lifestyle-related diseases. However, the entire digestive tract hormone has not yet been clarified, and there are many unclear parts.
そこで本発明は、これまでに知られていない消化管由来のタンパク質をコードする遺伝子の探索を行い、見出された遺伝子をもとに新たな医薬用途を提供することを目的とする。 Therefore, an object of the present invention is to search for a gene encoding a protein derived from a digestive tract that has not been known so far, and to provide a new pharmaceutical use based on the found gene.
本発明者らは、上記の点に鑑みて鋭意研究を重ねた結果、SST法(Signal Sequence Trap法：Nat Biotechnol. 1999 May;17(5):487-90. A signal sequence trap based on a constitutively active cytokine receptor. Kojima T, Kitamura T.)を採用してマウスの腸管から単離したcDNA断片をもとに取得したクローンCF266(mCF266)が腸管において特異的な発現をしていること、このmCF266の培養上清にはインスリン分泌誘導作用があること、アデノウイルスベクターを使用した強制発現系においてmCF266がin vivoでインスリンの分泌誘導作用を示すことを見出した。 The present inventors have conducted extensive studies in view of the above points, and as a result, the SST method (Signal Sequence Trap method: Nat Biotechnol. 1999 May; 17 (5): 487-90. A signal sequence trap based on a constitutively active cytokine receptor. Kojima T, Kitamura T.) clone CF266 (mCF266) obtained from a cDNA fragment isolated from the intestinal tract of a mouse is specifically expressed in the intestinal tract. It was found that mCF266 exhibits an insulin secretion-inducing action in vivo in a forced expression system using an adenovirus vector.
上記の知見に基づいてなされた本発明のインスリン分泌誘導剤は、請求項１記載の通り、次の（ａ）〜（ｃ）の少なくともいずれか１つを有効成分とすることを特徴とする；
（ａ）配列番号１又は配列番号４に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号１又は配列番号４に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列において１若しくは数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、かつ、インスリン分泌誘導作用を持つポリペプチド、
（ｃ）インスリン分泌誘導作用を持つ（ａ）又は（ｂ）のポリペプチドのフラグメント。
また、請求項２記載のインスリン分泌誘導剤は、請求項１記載のインスリン分泌誘導剤において、配列番号４に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列において１若しくは数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、かつ、インスリン分泌誘導作用を持つポリペプチドが配列番号７に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列からなるポリペプチドであることを特徴とする。
また、本発明のインスリン分泌誘導剤は、請求項３記載の通り、次の（ｄ）〜（ｆ）の少なくともいずれか１つを有効成分とすることを特徴とする；
（ｄ）配列番号３又は配列番号６に記載のアミノ酸配列からなるポリペプチド、
（ｅ）配列番号３又は配列番号６に記載のアミノ酸配列において１若しくは数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、かつ、インスリン分泌誘導作用を持つポリペプチド、
（ｆ）インスリン分泌誘導作用を持つ（ｄ）又は（ｅ）のポリペプチドのフラグメント。
また、請求項４記載のインスリン分泌誘導剤は、請求項３記載のインスリン分泌誘導剤において、配列番号６に記載のアミノ酸配列において１若しくは数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、かつ、インスリン分泌誘導作用を持つポリペプチドが配列番号９に記載のアミノ酸配列からなるポリペプチドであることを特徴とする。
また、請求項５記載のインスリン分泌誘導剤は、請求項３記載のインスリン分泌誘導剤において、インスリン分泌誘導作用を持つポリペプチドのフラグメントが配列番号１０に記載のアミノ酸配列を少なくとも含むポリペプチドであることを特徴とする。
また、請求項６記載のインスリン分泌誘導剤は、請求項３記載のインスリン分泌誘導剤において、インスリン分泌誘導作用を持つポリペプチドのフラグメントが配列番号１１に記載のアミノ酸配列を少なくとも含むポリペプチドであることを特徴とする。
また、請求項７記載のインスリン分泌誘導剤は、請求項３記載のインスリン分泌誘導剤において、インスリン分泌誘導作用を持つポリペプチドのフラグメントが配列番号１２に記載のアミノ酸配列を少なくとも含むポリペプチドであることを特徴とする。
また、本発明のインスリン分泌誘導組成物は、請求項８記載の通り、配列番号３に記載のアミノ酸配列からなるポリペプチド、配列番号６に記載のアミノ酸配列からなるポリペプチド、配列番号９に記載のアミノ酸配列からなるポリペプチド、配列番号１０に記載のアミノ酸配列を少なくとも含むポリペプチド、配列番号１１に記載のアミノ酸配列を少なくとも含むポリペプチド、配列番号１２に記載のアミノ酸配列を少なくとも含むポリペプチド、の少なくともいずれか１つを有効成分として含有することを特徴とする。
The insulin secretion inducer of the present invention made based on the above findings is characterized in that, as described in claim 1, at least one of the following (a) to (c) is an active ingredient;
(A) a polypeptide consisting of the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4,
In the amino acid sequence encoded by DNA comprising the nucleotide sequence of (b) SEQ ID NO: 1 or SEQ ID NO: 4 wherein one or a few amino acids are substituted, deleted and / or added in the amino acid sequence, and insulin A polypeptide having a secretion-inducing action,
(C) A fragment of the polypeptide (a) or (b) having an insulin secretion-inducing action.
Further, the insulin secretion-inducing agent according to claim 2, wherein, in the insulin secretion-inducing agent according to claim 1 wherein, one or several amino acids in the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4 is substituted , deletion and / or added in the amino acid sequence, and the polypeptide having the insulin secretion-inducing activity is a polypeptide consisting of the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 7 Features.
Moreover, the insulin secretion inducer of the present invention is characterized in that, as described in claim 3, at least one of the following (d) to (f) is an active ingredient;
(D) a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6,
(E) 1 or several amino acids in the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 6 is substituted, deleted and / or added in the amino acid sequence, and polypeptides having insulin secretion inducing action,
(F) A fragment of the polypeptide (d) or (e) having an insulin secretion-inducing action.
The insulin secretion inducer according to claim 4 is the insulin secretion inducer according to claim 3, wherein one or several amino acids are substituted, deleted, and / or added in the amino acid sequence of SEQ ID NO: 6. to the amino acid sequence, and wherein the polypeptide having insulin secretion-inducing activity is a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 9.
The insulin secretion inducer according to claim 5 is a polypeptide in which the fragment of a polypeptide having an insulin secretion induction action in the insulin secretion inducer according to claim 3 includes at least the amino acid sequence of SEQ ID NO: 10. It is characterized by that.
The insulin secretion inducer according to claim 6 is a polypeptide in which the fragment of the polypeptide having an insulin secretion induction action in the insulin secretion inducer according to claim 3 includes at least the amino acid sequence set forth in SEQ ID NO: 11. It is characterized by that.
The insulin secretion inducer according to claim 7 is a polypeptide in which the fragment of a polypeptide having an insulin secretion induction action in the insulin secretion inducer according to claim 3 includes at least the amino acid sequence of SEQ ID NO: 12. It is characterized by that.
Further, insulin secretion inducing composition of the present invention, as claimed in claim 8, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3, a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 9 a polypeptide consisting of an amino acid sequence at least comprising a polypeptide the amino acid sequence set forth in SEQ ID NO: 10, at least comprising a polypeptide the amino acid sequence set forth in SEQ ID NO: 11, at least comprising a polypeptide the amino acid sequence set forth in SEQ ID NO: 12, It contains at least any one of these as an active ingredient .
本発明によれば、糖尿病をはじめとする種々の代謝性疾患の治療に有効なインスリン分泌誘導剤及びインスリン分泌誘導組成物を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the insulin secretion inducer and insulin secretion induction composition which are effective in the treatment of various metabolic diseases including diabetes can be provided.
本発明のインスリン分泌誘導剤は、次の（ａ）〜（ｃ）の少なくともいずれか１つを有効成分とすることを特徴とするものである；
（ａ）配列番号１又は配列番号４に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号１又は配列番号４に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列において１若しくは数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、かつ、インスリン分泌誘導作用を持つポリペプチド、
（ｃ）インスリン分泌誘導作用を持つ（ａ）又は（ｂ）のポリペプチドのフラグメント。
The insulin secretion inducer of the present invention is characterized in that it comprises at least one of the following (a) to (c) as an active ingredient;
(A) a polypeptide consisting of the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4,
In the amino acid sequence encoded by DNA comprising the nucleotide sequence of (b) SEQ ID NO: 1 or SEQ ID NO: 4 wherein one or a few amino acids are substituted, deleted and / or added in the amino acid sequence, and insulin A polypeptide having a secretion-inducing action,
(C) A fragment of the polypeptide (a) or (b) having an insulin secretion-inducing action.
本発明において、本発明者らがマウスの腸管から取得したｍＣＦ２６６は配列番号１に記載の塩基配列からなるＤＮＡであるが、このＤＮＡはマウスの筋肉に発現している膜タンパクＴｍ４ｓｆ２０（Ｔｒａｎｓｍｅｍｂｒａｎｅ ４ Ｌ ｓｉｘ ｆａｍｉｌｙ ｍｅｍｂｅｒ ２０）をコードするＤＮＡとして公知の全長１５０５ｂｐのものである（ＮＣＢＩ：ＬＯＣＵＳ ＮＭ ０２５４５３）。ｍＣＦ２６６のＣＤＳは４１．．７２１であり、配列番号３に記載の２２６個のアミノ酸配列からなるポリペプチドをコードしている（配列番号２参照）。しかしながら、このポリペプチドがインスリン分泌誘導作用を持つことについての報告はこれまでのところ存在しない。また、本発明者らは、ｍＣＦ２６６に対応するヒトＣＦ２６６（ｈＣＦ２６６）についてもｍＣＦ２６６と同様の作用を有することを確認している。ｈＣＦ２６６は配列番号４に記載の塩基配列からなるＤＮＡであり、ヒトＴＭ４ＳＦ２０をコードするＤＮＡとして公知の全長２３０８ｂｐのものである（ＮＣＢＩ：ＬＯＣＵＳ ＮＭ ０２４７９５）。ｈＣＦ２６６のＣＤＳは３８．．７２７であり、配列番号６に記載の２２９個のアミノ酸配列からなるポリペプチドをコードしている（配列番号５参照）。しかしながら、このポリペプチドがインスリン分泌誘導作用を持つことについての報告もまたこれまでのところ存在しない。 In the present invention, mCF266 obtained from the intestinal tract of the mouse by the present inventors is a DNA having the base sequence set forth in SEQ ID NO: 1, and this DNA is a membrane protein Tm4sf20 (Transmembrane 4 L expressed in the muscle of the mouse. The DNA encoding six family members 20) has a known length of 1505 bp (NCBI: LOCUS NM 025453). The CDS of mCF266 is 41. . 721 and encodes a polypeptide consisting of the 226 amino acid sequence set forth in SEQ ID NO: 3 (see SEQ ID NO: 2). However, there has been no report so far regarding that this polypeptide has an insulin secretion-inducing action. The present inventors have also confirmed that human CF266 (hCF266) corresponding to mCF266 has the same action as mCF266. hCF266 is a DNA having the nucleotide sequence set forth in SEQ ID NO: 4, and has a total length of 2308 bp known as DNA encoding human TM4SF20 (NCBI: LOCUS NM 024795). The CDS of hCF266 is 38. . 720, and encodes a polypeptide consisting of the 229 amino acid sequence set forth in SEQ ID NO: 6 (see SEQ ID NO: 5). However, there has been no report so far that this polypeptide has an insulin secretion-inducing action.
配列番号４に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列において１若しくは数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、かつ、インスリン分泌誘導作用を持つポリペプチドとしては、例えば、ｈＣＦ２６６の一塩基多型（ＳＮＰｓ）に基づく配列番号７に記載の塩基配列からなるＤＮＡによりコードされる配列番号９に記載の２２９個のアミノ酸配列からなるポリペプチドが挙げられる（配列番号８参照）。配列番号６に記載のアミノ酸配列からなるポリペプチドと、配列番号９に記載のアミノ酸配列からなるポリペプチドの相違は、２７番目のアミノ酸が前者がバリン（ｈＣＦ２６６（２７Ｖ））であるのに対して後者がアラニン（ｈＣＦ２６６（２７Ａ））であることである。 One or several amino acids in the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 4 is substituted, deleted and / or added in the amino acid sequence, and polypeptides having insulin secretion inducing action Examples of the polypeptide include a polypeptide consisting of 229 amino acid sequences of SEQ ID NO: 9 encoded by DNA consisting of the nucleotide sequence of SEQ ID NO: 7 based on the single nucleotide polymorphisms (SNPs) of hCF266 ( See SEQ ID NO: 8). The difference between the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 is that the 27th amino acid is valine (hCF266 (27V)) in the former. The latter is alanine (hCF266 (27A)).
インスリン分泌誘導作用を持つポリペプチドのフラグメントとしては、例えば、配列番号６に記載のアミノ酸配列からなるポリペプチドの９８〜１１６番目に相当する１９個のアミノ酸配列ＡＬＹＣＭＬＩＳＩＱＡＬＬＫＧＰＬＭＣ（配列番号１０）を少なくとも含むポリペプチド、同７８〜９６番目に相当する１９個のアミノ酸配列ＣＮＮＲＴＧＭＦＬＳＳＬＦＳＶＩＴＶＩ（配列番号１１）を少なくとも含むポリペプチド、同１６１から１７９番目に相当する１９個のアミノ酸配列ＴＳＮＤＴＭＡＳＧＷＲＡＳＳＦＨＦＤＳ（配列番号１２）を少なくとも含むポリペプチドが挙げられる。 Examples of the polypeptide fragment having an insulin secretion-inducing action include a polypeptide containing at least the 19 amino acid sequence ALYCMLISIQALLKGPLMC (SEQ ID NO: 10) corresponding to the 98th to 116th positions of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6. Peptide, Polypeptide including at least 19 amino acid sequence CNNNRTGMFLSSLFSVVIVI (SEQ ID NO: 11) corresponding to positions 78 to 96, Poly including at least 19 amino acid sequence TSNDTMASGWRASSFHFDS (SEQ ID NO: 12) corresponding to positions 161 to 179 Peptides are mentioned.
本発明のインスリン分泌誘導剤の有効成分となるポリペプチドやそのフラグメントは、例えば、自体公知の方法で配列番号１や配列番号４や配列番号７に記載の塩基配列からなるＤＮＡを外来遺伝子として胎児腎臓上皮細胞であるＨＥＫ２９３細胞やＨＥＫ２９３Ｔ細胞の他、ＣＨＯ−Ｋ１細胞に例示される動物細胞などを宿主細胞として組み込み、当該細胞を培養して遺伝子発現させることで、その培養上清から得ることができるが、化学合成により製造してもよい。これらの分離・精製は、例えば、イオン交換樹脂、分配クロマトグラフィー、ゲルクロマトグラフィー、逆相クロマトグラフィーなどのペプチド化学において通常使用される方法によって行うことができる。 Polypeptide or fragment thereof as the active ingredient of the insulin secretion-inducing agent of the present invention, for example, fetal a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 4 or SEQ ID NO: 7 in a manner known per se as a foreign gene In addition to HEK293 cells and HEK293T cells, which are kidney epithelial cells, animal cells exemplified by CHO-K1 cells are incorporated as host cells, and the cells are cultured and gene expression is performed to obtain them from the culture supernatant. It can be produced by chemical synthesis. These separations and purifications can be performed by methods usually used in peptide chemistry such as ion exchange resin, partition chromatography, gel chromatography, and reverse phase chromatography.
本発明のインスリン分泌誘導剤は、例えば、その有効成分となるポリペプチドやそのフラグメントを必要に応じて自体公知の方法で薬学的に許容される塩酸塩などの無機酸塩、酢酸塩などの有機酸塩、ナトリウム塩などのアルカリ金属塩などに変換した上で凍結乾燥品として製剤化し、必要時に生理食塩水などに溶解して注射剤の形態で静脈内投与することでインスリンの分泌を誘導することができる。その用法用量は患者の性別、年齢、体重、病態などによって適宜決定すればよい。なお、本発明のインスリン分泌誘導剤の有効成分となるポリペプチドやそのフラグメントは、高度に精製された純品を単独で使用してもよいし、複数種類を混合して使用してもよく、種々のインスリン分泌誘導組成物の形態で使用することができる。 The insulin secretion-inducing agent of the present invention includes, for example, a polypeptide or fragment thereof as an active ingredient, if necessary, a pharmaceutically acceptable inorganic acid salt such as hydrochloride, an organic acid salt such as acetate, and the like. Insulin secretion is induced by converting to acid salts, alkali metal salts such as sodium salts, etc., formulating as freeze-dried products, and dissolving in physiological saline etc. when necessary and administering intravenously in the form of injections be able to. The dosage may be appropriately determined according to the patient's sex, age, weight, disease state, and the like. In addition, as a polypeptide or fragment thereof as an active ingredient of the insulin secretion inducer of the present invention, a highly purified pure product may be used alone, or a plurality of types may be used in combination. It can be used in the form of various insulin secretion-inducing compositions.
また、本発明の説明に供する、遺伝子治療によりインスリン分泌誘導を行うためのウイルスベクターは、外来遺伝子の発現が可能なウイルスベクターに外来遺伝子として配列番号１、配列番号４、配列番号７のいずれかに記載の塩基配列からなるＤＮＡ又はこれらのＤＮＡとストリンジェントな条件下でハイブリダイズするＤＮＡを組み込んでなることを特徴とするものである。ここで「ストリンジェントな条件下でハイブリダイズするＤＮＡ」とは、対象とするＤＮＡをプローブとして使用し、コロニーハイブリダイゼーション法、プラークハイブリダイゼーション法、サザンブロットハイブリダイゼーション法などを採用することにより取得できるＤＮＡを意味し、例えば、コロニーやプラーク由来のＤＮＡを固定化したフィルタを使用し、０．７〜１．０Ｍ塩化ナトリウム存在下６５℃でハイブリダイゼーションを行った後、０．１〜２×ＳＳＣ溶液（１×ＳＳＣの組成：１５０ｍＭ塩化ナトリウム、１５ｍＭクエン酸ナトリウム）を使用し、６５℃条件下でフィルタを洗浄することにより同定できるＤＮＡなどが挙げられる（必要であれば例えばＭｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， ２ｎｄ Ｅｄ．， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ， Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ， ＮＹ．， １９８９．などを参照のこと）。ストリンジェントな条件下でハイブリダイズするＤＮＡの塩基配列の、プローブとして使用するＤＮＡの塩基配列との相同性は、８０％以上が好ましく、９０％以上がより好ましく、９３％以上がさらに好ましく、９５％以上が特に好ましく、９８％以上が最も好ましい。本発明の説明に供する、遺伝子治療によりインスリン分泌誘導を行うためのウイルスベクターの具体例としては、自体公知の方法で配列番号１、配列番号４、配列番号７のいずれかに記載の塩基配列からなるＤＮＡをＣＡＧプロモーターなどに連結して構築したアデノウイルスベクターなどが挙げられ、このようなウイルスベクターは静脈内投与することで体内において優れたインスリン分泌誘導作用を示す。 In addition, a viral vector for inducing insulin secretion by gene therapy used for the explanation of the present invention is any one of SEQ ID NO: 1, SEQ ID NO: 4, and SEQ ID NO: 7 as a foreign gene on a viral vector capable of expressing a foreign gene. those characterized by comprising incorporating a DNA hybridizing with DNA or these DNA under stringent conditions comprising the nucleotide sequence of. Here, “DNA that hybridizes under stringent conditions” can be obtained by using a target DNA as a probe and adopting a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. It means DNA, for example, using a filter on which colony or plaque-derived DNA is immobilized, hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M sodium chloride, and then 0.1 to 2 × SSC. Examples include DNA that can be identified by using a solution (composition of 1 × SSC: 150 mM sodium chloride, 15 mM sodium citrate) and washing the filter at 65 ° C. (for example, Molecular Cloning: A Laboratory, if necessary). anual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., see, such as 1989.). The homology of the base sequence of the DNA that hybridizes under stringent conditions with the base sequence of the DNA used as the probe is preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, 95 % Or more is particularly preferable, and 98% or more is most preferable. Explaining the present invention, specific examples of viral vectors for performing insulin secretion induced by gene therapy, SEQ ID NO: 1 in a manner known per se, SEQ ID NO: 4, the nucleotide sequence of any one of SEQ ID NO: 7 The adenovirus vector constructed by linking the DNA to a CAG promoter or the like can be mentioned, and such a virus vector exhibits an excellent insulin secretion-inducing action in the body when administered intravenously.
以下、本発明を実施例によって詳細に説明するが、本発明は以下の記載によって何ら限定して解釈されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited and interpreted by the following description at all.
参考例１：ｍＣＦ２６６が腸管に特異的に発現していることの確認
ＳＳＴ法を採用してマウスの腸管から取得したｍＣＦ２６６のｍＲＮＡの臓器発現分布をノザンブロット法により検討を行った。具体的には、マウスより各臓器を摘出し、ＴＲＩｚｏｌ（ｉｎｖｉｔｒｏｇｅｎ）により添付の説明書に従いｔｏｔａｌ ＲＮＡを抽出し、定法に従い１０μｇ／ｌａｎｅとなるようにメンブレンを作製し、［α−３２Ｐ］ｄＣＴＰを使用して標識したｍＣＦ２６６のｃＤＮＡ（ｍＲＮＡクローン）をプローブにしてハイブリダイズすることで行った。その結果を図１に示す。図１から明らかなように、ｍＣＦ２６６は小腸（Ｉｎｔｅｓｔｉｎｅ）において特異的な発現をしていることがわかった。
Reference Example 1: Confirmation that mCF266 is specifically expressed in the intestinal tract The organ expression distribution of mCF266 mRNA obtained from the intestinal tract of mice using the SST method was examined by Northern blotting. Specifically, each organ was removed from the mouse, total RNA was extracted according to the attached instructions using TRIzol (invitrogen), and a membrane was prepared according to a standard method to 10 μg / lane, and [α- 32 P This was carried out by hybridizing using mCF266 cDNA (mRNA clone) labeled with dCTP as a probe. The result is shown in FIG. As is clear from FIG. 1, mCF266 was found to be specifically expressed in the small intestine (Intestine).
実施例１：マウス膵β細胞由来培養細胞株ＭＩＮ６細胞に対するＣＦ２６６の培養上清のインスリン分泌誘導作用
１０ｃｍ ｄｉｓｈに５％ＦＣＳと抗生物質（ペニシリン１００Ｕ／ｍＬ、ストレプトマイシン１０ｍｇ／ｍＬ）を添加したＤＭＥＭ培地で継代したＨＥＫ２９３Ｔ細胞を１×１０６撒き、その翌日にｍＣＦ２６６発現ベクター（ｐＣＡＧＧＳ−ｍＣＦ２６６）をＦｕＧＥＮＥ６（Ｒｏｃｈｅ）を使用してＨＥＫ２９３Ｔ細胞にトランスフェクトしてｍＣＦ２６６を強制発現させ、その２４ｈ後にＯｐｔｉ−ＭＥＭ培地に交換し、さらにその２４ｈ後に培養上清を回収した。１５％ＦＣＳと抗生物質（ペニシリン１００Ｕ／ｍＬ、ストレプトマイシン１０ｍｇ／ｍＬ）と２−メルカプトエタノールを添加したＤＭＥＭ培地（Ｈｉｇｈ Ｇｌｕｃｏｓｅ、ＧＩＢＣＯ；現Ｉｎｖｉｔｒｏｇｅｎ）で継代したＭＩＮ６細胞を２４ｗｅｌｌプレートに３×１０５／ｗｅｌｌ撒き、その翌日、ＫＲＢＨ緩衝液（２．８ｍＭグルコース）を０．５ｍＬ／ｗｅｌｌ添加して３０分間前培養した後、上記の培養上清とＫＲＢＨ緩衝液との混合液（１：１（ｖ／ｖ））を０．５ｍＬ／ｗｅｌｌ添加して１時間刺激を行い、ＭＩＮ６細胞のグルコース応答性のインスリン分泌（ＧＳＩＳ）を測定した。測定は培養液中のインスリンをＥＬＩＳＡ法（シバヤギのレビスインスリンキットを使用：以下同じ）で測定することで行った。なお、インスリン値はＭＩＮ６細胞の総タンパクで補正した。その結果を、上記の方法と同様の方法で空ベクター（ｐＣＡＧＧＳ）をＨＥＫ２９３Ｔ細胞にトランスフェクトして得た培養上清を添加した場合の結果（Ｍｏｃｋ）と、ヒト腸管ｃＤＮＡライブラリー（クローンテック；現ＢＤ Ｂｉｏｓｃｉｅｎｃｅｓ）からクローニングした配列番号４に記載の塩基配列からなるＤＮＡ（ｈＣＦ２６６（２７Ｖ））と公知の方法でＤＮＡ（ｈＣＦ２６６（２７Ｖ））に対して変異導入することで取得した配列番号７に記載の塩基配列からなるＤＮＡ（ｈＣＦ２６６（２７Ａ））のそれぞれの発現ベクターをＨＥＫ２９３Ｔ細胞にトランスフェクトしてそれぞれを強制発現させて得た培養上清を添加した場合の結果とともに図２に示す。図２から明らかなように、ＣＦ２６６を強制発現させた培養上清はＭＩＮ６細胞に対してグルコース応答性のインスリン分泌を誘導し、その作用はとりわけｈＣＦ２６６（２７Ｖ）を強制発現させた場合が優れていた。
Example 1: Insulin secretion-inducing action of culture supernatant of CF266 on mouse pancreatic β cell-derived cultured cell line MIN6 cells DMEM medium supplemented with 10% dish with 5% FCS and antibiotics (penicillin 100 U / mL, streptomycin 10 mg / mL) HEK293T cells passaged in 1) were seeded at 1 × 10 6 , and the next day, mCF266 expression vector (pCAGGS-mCF266) was transfected into HEK293T cells using FuGENE6 (Roche), and mCF266 was forcibly expressed 24 hours later. -It replaced | exchanged for the MEM culture medium and also culture | cultivation supernatant was collect | recovered 24 hours after that. MIN6 cells passaged in DMEM medium (High Glucose, GIBCO; currently Invitrogen) supplemented with 15% FCS, antibiotics (penicillin 100 U / mL, streptomycin 10 mg / mL) and 2-mercaptoethanol were added to 3 × 10 5 cells on a 24 well plate. / Well, the next day, after adding 0.5 mL / well of KRBH buffer (2.8 mM glucose) and pre-incubating for 30 minutes, a mixture of the above culture supernatant and KRBH buffer (1: 1 ( v / v)) was added at 0.5 mL / well for stimulation for 1 hour, and glucose-responsive insulin secretion (GSIS) of MIN6 cells was measured. The measurement was carried out by measuring insulin in the culture solution by ELISA (using Shiba Goat's Levis Insulin Kit: the same applies hereinafter). The insulin level was corrected with the total protein of MIN6 cells. The results are as follows (Mock) when a culture supernatant obtained by transfecting HEK293T cells with an empty vector (pCAGGS) in the same manner as described above, and a human intestinal cDNA library (Clontech; in SEQ ID NO: 7 obtained by mutagenesis with respect to DNA (hCF266 (27V)) and in known manner DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 was cloned from the current BD Biosciences) (hCF266 (27V) ) together with the results of the case of adding the culture supernatant obtained by forced expression of each respective expression vector was transfected into HEK293T cells DNA comprising the nucleotide sequence of (hCF266 (27A)) shown in FIG. As is apparent from FIG. 2, the culture supernatant in which CF266 was forcibly expressed induced glucose-responsive insulin secretion to MIN6 cells, and the action was particularly excellent when hCF266 (27V) was forcibly expressed. It was.
参考例２：ｍＣＦ２６６のｉｎ ｖｉｖｏでのインスリン分泌誘導作用
ＣＡＧプロモーターにｍＣＦ２６６を連結して構築したアデノウイルスベクター（Ｉｎｖｉｔｒｏｇｅｎの商品名ＶｉｒａＰｏｗｅｒを使用して作製）を、高脂肪高ショ糖負荷食を与えた糖尿病モデルマウスＫＫ／Ａｙマウスに５×１０９ＰＦＵ静脈内投与した。その４日後、静脈内糖負荷試験（ｉ．ｖ．ＧＴＴ）を行い、経時的に採血し、血清中の血糖値、インスリン値を測定した。その結果を図３に示す。図３から明らかなように、糖尿病モデルマウスの体内でｍＣＦ２６６を発現させると、対照としてＧＦＰを発現させた場合と比較して、血糖値は低い傾向が見られるに過ぎなかったが（図３左）、インスリン値は明らかに高く、ｍＣＦ２６６の発現はインスリンの分泌を誘導することがわかった（図３右）。
Reference Example 2: In vivo insulin secretion inducing action of mCF266 An adenoviral vector constructed by linking mCF266 to the CAG promoter (made using Invitrogen's trade name ViraPower) was fed with a high-fat, high-sucrose-loaded diet. 5 × 10 9 PFU was intravenously administered to diabetic model mice KK / Ay mice. Four days later, an intravenous glucose tolerance test (iv GTT) was performed, blood was collected over time, and blood glucose levels and insulin levels in the serum were measured. The result is shown in FIG. As is apparent from FIG. 3, when mCF266 was expressed in the body of a diabetes model mouse, the blood glucose level tended to be lower than when GFP was expressed as a control (FIG. 3 left). ), Insulin levels were clearly high, and mCF266 expression was found to induce insulin secretion (right in FIG. 3).
実施例２：マウス膵臓ランゲルハンス氏島細胞に対するｈＣＦ２６６（２７Ｖ）の培養上清に含まれるポリペプチドのインスリン分泌誘導作用
実施例１に記載の方法でｈＣＦ２６６（２７Ｖ）の発現ベクターをＨＥＫ２９３Ｔ細胞にトランスフェクトしてｈＣＦ２６６（２７Ｖ）を強制発現させて得た培養上清を陰イオン交換カラム（ＰＯＲＯＳ ＨＱカラム、ＡＢＩ）を使用して分画した。マウスから単離した膵臓ランゲルハンス氏島細胞を２４ｗｅｌｌプレートに１０ｉｓｌｅｔｓ／ｗｅｌｌ撒き、ＲＰＭＩ１６４０培地（１０％ＦＣＳ、ＧＩＢＣＯ；現Ｉｎｖｉｔｒｏｇｅｎ）中で２時間培養した後、ＫＲＢＨ緩衝液（２．８ｍＭグルコース）を０．５ｍＬ／ｗｅｌｌ添加して３０分間前培養した後、上記の培養上清画分とＫＲＢＨ緩衝液との混合液（１：１（ｖ／ｖ））を０．５ｍＬ／ｗｅｌｌ添加して１時間刺激を行い、マウス膵臓ランゲルハンス氏島細胞のグルコース応答性のインスリン分泌（ＧＳＩＳ）を測定した。測定は培養液中のインスリンをＥＬＩＳＡ法で測定することで行った。インスリン分泌誘導活性が認められた画分を質量分析装置（ＴＯＦ−ＭＡＳ）で解析し、特異的に見られたピークの質量から予測された１９個のアミノ酸配列からなるポリペプチド、ＡＬＹＣＭＬＩＳＩＱＡＬＬＫＧＰＬＭＣ（ポリペプチドＡ：配列番号１０）、ＣＮＮＲＴＧＭＦＬＳＳＬＦＳＶＩＴＶＩ（ポリペプチドＢ：配列番号１１）、ＴＳＮＤＴＭＡＳＧＷＲＡＳＳＦＨＦＤＳ（ポリペプチドＣ：配列番号１２）を化学合成した。これら３種類のポリペプチドＡ〜ＣのそれぞれをＫＲＢＨ緩衝液に１０ｎＭの濃度で溶解した溶液を、上記の方法と同様の方法で３０分間前培養したマウス膵臓ランゲルハンス氏島細胞に０．５ｍＬ／ｗｅｌｌ添加して３０分間刺激を行い、上記の方法と同様の方法でマウス膵臓ランゲルハンス氏島細胞のグルコース応答性のインスリン分泌を測定した。なお、インスリン値はマウス膵臓ランゲルハンス氏島細胞の総ＤＮＡで補正した。その結果を、ＫＲＢＨ緩衝液のみを添加して刺激を行ったこと以外は上記の方法と同様の方法で実験を行った場合の結果、ｈＣＦ２６６（２７Ｖ）の培養上清とＫＲＢＨ緩衝液との混合液（１：１（ｖ／ｖ））を添加して刺激を行ったこと以外は上記の方法と同様の方法で実験を行った場合の結果、ヒトＧＬＰ−１（ペプチド研究所）をＫＲＢＨ緩衝液に１０ｎＭの濃度で溶解した溶液を添加して刺激を行ったこと以外は上記の方法と同様の方法で実験を行った場合の結果とともに図４に示す。また、ＫＲＢＨ緩衝液（２．８ｍＭグルコース）に代えてＫＲＢＨ緩衝液（２０ｍＭグルコース）を使用して実験を行った場合の結果を図４にあわせて示す。図４から明らかなように、配列番号４に記載の塩基配列からなるｈＣＦ２６６（２７Ｖ）によりコードされる配列番号６に記載のアミノ酸配列からなるポリペプチドのフラグメントである３種類のポリペプチドＡ〜Ｃは、マウス膵臓ランゲルハンス氏島細胞に対してグルコース応答性のインスリン分泌を誘導することがわかった。
Example 2 : Insulin secretion-inducing action of polypeptide contained in culture supernatant of hCF266 (27V) on mouse pancreatic Langerhans islet cells Transfected with an expression vector of hCF266 (27V) in HEK293T cells by the method described in Example 1 The culture supernatant obtained by forcibly expressing hCF266 (27V) was fractionated using an anion exchange column (POROS HQ column, ABI). Pancreatic Langerhans islet cells isolated from mice were seeded on 24 well plates at 10 islets / well, cultured in RPMI 1640 medium (10% FCS, GIBCO; current Invitrogen) for 2 hours, and then KRBH buffer (2.8 mM glucose) was added to 0. After adding 5 mL / well and pre-incubating for 30 minutes, 0.5 mL / well of the mixed solution of the above culture supernatant fraction and KRBH buffer (1: 1 (v / v)) was added for 1 hour. Stimulation was performed and glucose-responsive insulin secretion (GSIS) of mouse pancreatic islets was measured. The measurement was performed by measuring insulin in the culture solution by ELISA. Polypeptide fractions insulin secretion-inducing activity was observed and analyzed by mass spectrometry (TOF-MAS), consisting of the predicted 19 amino acid sequence from the mass of specifically seen peaks, ALYCMLISIQALLKGPLMC (polypeptide A: SEQ ID NO: 10), CNNNRTGMFLSSLFSVITVI (polypeptide B: SEQ ID NO: 11), and TSNDTMASGWRASSFHFDS (polypeptide C: SEQ ID NO: 12) were chemically synthesized. A solution prepared by dissolving each of these three polypeptides A to C in KRBH buffer at a concentration of 10 nM was added to 0.5 mL / well of mouse pancreatic Langerhans islet cells pre-cultured for 30 minutes in the same manner as described above. After addition, stimulation was performed for 30 minutes, and glucose-responsive insulin secretion of mouse pancreatic Langerhans islet cells was measured by the same method as described above. The insulin level was corrected with the total DNA of mouse pancreatic Langerhans islet cells. As a result of the experiment conducted in the same manner as the above method except that only the KRBH buffer solution was added for stimulation, mixing of the hCF266 (27V) culture supernatant and the KRBH buffer solution was performed. As a result of the experiment conducted by the same method as described above except that the solution (1: 1 (v / v)) was added to stimulate the human GLP-1 (Peptide Institute) FIG. 4 shows the results when the experiment was conducted in the same manner as described above except that the solution was dissolved in a solution of 10 nM and stimulated. Moreover, it replaces with a KRBH buffer solution (2.8 mM glucose), and the result at the time of experimenting using a KRBH buffer solution (20 mM glucose) is shown according to FIG. As is clear from FIG. 4, three types of polypeptides A to C, which are fragments of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 encoded by hCF266 (27V) consisting of the base sequence shown in SEQ ID NO: 4, Was found to induce glucose-responsive insulin secretion on mouse pancreatic islet cells.
製剤例１：
自体公知の方法で配列番号１０に記載のアミノ酸配列からなるポリペプチドを滅菌してから凍結乾燥することで静脈注射剤として製剤化した。
Formulation Example 1:
The polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10 was sterilized by a method known per se, and then lyophilized to prepare an intravenous injection.
本発明は、糖尿病をはじめとする種々の代謝性疾患の治療に有効なインスリン分泌誘導剤及びインスリン分泌誘導組成物を提供することができる点において産業上の利用可能性を有する。 INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide an insulin secretion inducer and an insulin secretion induction composition that are effective in the treatment of various metabolic diseases including diabetes.
Claims (8)
（ａ）配列番号１又は配列番号４に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列からなるポリペプチド、
（ｂ）配列番号１又は配列番号４に記載の塩基配列からなるＤＮＡによりコードされるアミノ酸配列において１若しくは数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、かつ、インスリン分泌誘導作用を持つポリペプチド、
（ｃ）インスリン分泌誘導作用を持つ（ａ）又は（ｂ）のポリペプチドのフラグメント。 An insulin secretion inducer comprising at least one of the following (a) to (c) as an active ingredient;
(A) a polypeptide consisting of the amino acid sequence encoded by the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4,
In the amino acid sequence encoded by DNA comprising the nucleotide sequence of (b) SEQ ID NO: 1 or SEQ ID NO: 4 wherein one or a few amino acids are substituted, deleted and / or added in the amino acid sequence, and insulin A polypeptide having a secretion-inducing action,
(C) A fragment of the polypeptide (a) or (b) having an insulin secretion-inducing action.
（ｄ）配列番号３又は配列番号６に記載のアミノ酸配列からなるポリペプチド、
（ｅ）配列番号３又は配列番号６に記載のアミノ酸配列において１若しくは数個のアミノ酸が置換、欠失及び／又は付加されたアミノ酸配列からなり、かつ、インスリン分泌誘導作用を持つポリペプチド、
（ｆ）インスリン分泌誘導作用を持つ（ｄ）又は（ｅ）のポリペプチドのフラグメント。 An insulin secretion inducer comprising at least one of the following (d) to (f) as an active ingredient;
(D) a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 6,
(E) 1 or several amino acids in the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 6 is substituted, deleted and / or added in the amino acid sequence, and polypeptides having insulin secretion inducing action,
(F) A fragment of the polypeptide (d) or (e) having an insulin secretion-inducing action.
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