JP2010057451A - NUCLEIC ACID MOLECULE HAVING BINDING PROPERTY TO MOUSE-DERIVED IgG ANTIBODY AND DETECTION KIT - Google Patents
NUCLEIC ACID MOLECULE HAVING BINDING PROPERTY TO MOUSE-DERIVED IgG ANTIBODY AND DETECTION KIT Download PDFInfo
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Abstract
【課題】抗体よりも簡便に調製可能で、且つ抗体と比べて同等以上の結合性を有する、マウス由来のＩｇＧ抗体に結合性を有する核酸分子、及びこの核酸分子を用いた検出キットを提供する。
【解決手段】マウス由来のＩｇＧ抗体に結合性を有する核酸分子。マウス由来のＩｇＧ抗体への結合を検出する検出キット。上記の核酸分子を含有する試薬。
【選択図】なしProvided is a nucleic acid molecule that can be prepared more easily than an antibody and has a binding property equal to or higher than that of an antibody, and has a binding property to a mouse-derived IgG antibody, and a detection kit using the nucleic acid molecule. .
A nucleic acid molecule capable of binding to a mouse-derived IgG antibody. A detection kit for detecting binding to a mouse-derived IgG antibody. A reagent containing the above nucleic acid molecule.
[Selection figure] None
Description
本発明は、マウス由来のＩｇＧ抗体に結合性を有する核酸分子、及びこの抗体への結合を検出する検出キットに関する。 The present invention relates to a nucleic acid molecule capable of binding to a mouse-derived IgG antibody, and a detection kit for detecting binding to the antibody.
マウスや核酸分子等のオリゴヌクレオチドは、主としてタンパク質の合成に関与する分子種としての機能を主として有するものと考えられてきたが、リボザイムやＲＮＡｉといった、遺伝子がタンパク質や高分子等の分子種と直接相互作用することにより、分子種の有する機能を制御し得る現象が見出され、注目されている。なかでも、アプタマーは、近年、タンパク質などの分子種に結合して、その機能を改変し得る核酸として注目されており、医薬品等への応用を目的として、新規のアプタマーが多く取得されている。 Oligonucleotides such as mice and nucleic acid molecules have been considered to have mainly functions as molecular species involved in protein synthesis, but genes such as ribozymes and RNAi are directly related to molecular species such as proteins and macromolecules. Phenomena that can control the functions of molecular species by interacting with each other have been found and are attracting attention. Among these, aptamers have recently attracted attention as nucleic acids that can bind to molecular species such as proteins and modify their functions, and many new aptamers have been obtained for the purpose of application to pharmaceuticals and the like.
一方、マウス、ラット、ウサギなどの実験動物に由来するＩｇＧなどの各クラスに属する抗体は、タンパク質などの抗原や、抗原−抗体複合体を形成し得る抗体など、高分子化合物に水素結合などの結合様式で結合し得るタンパク性の物質を総称する。抗体は、例えば抗原−抗体複合体における抗体に特異的に結合する抗体として、診断薬など医療分野等において広く用いられている。 On the other hand, antibodies belonging to each class such as IgG derived from experimental animals such as mice, rats, and rabbits are capable of hydrogen bonding to polymer compounds such as antigens such as proteins and antibodies that can form antigen-antibody complexes. It is a generic term for proteinaceous substances that can bind in a binding mode. An antibody is widely used in the medical field such as a diagnostic drug as an antibody that specifically binds to an antibody in an antigen-antibody complex.
しかしながら、抗体は、抗原と特異的な活性を有する点で、有用であるものの、抗体の調製には、種々の問題が指摘されている。例えば、抗体を得るには、マウス、ラット、ウサギ等の被免疫動物に抗原を反復的に注入して免疫反応を惹起した後、血清等から、所望する、抗原との結合性を有する画分を調製する必要があり、作業の面でも、コストの面でも、非常に不利である。また、抗体は、この抗体が特異的に結合する抗原以外にも、種々のタンパク質や、ポリプロピレン（ＰＰ）、ポリエチレン（ＰＥ）といった容器等を構成する材料にも非特異的に結合する性質を有しており、ハンドリングの面でも不利である。さらに、抗体の調製には、上述の通り、被免疫動物を用いる必要があり、動物愛護の面からも、好ましいものではない。 However, although antibodies are useful in that they have specific activity with antigens, various problems have been pointed out in the preparation of antibodies. For example, in order to obtain an antibody, an antigen is repeatedly injected into an immunized animal such as a mouse, a rat, or a rabbit to induce an immune reaction, and then a desired fraction that has an antigen-binding property from serum or the like. This is very disadvantageous both in terms of work and cost. In addition to the antigen to which the antibody specifically binds, the antibody has the property of non-specifically binding to various proteins and materials constituting containers such as polypropylene (PP) and polyethylene (PE). This is also disadvantageous in terms of handling. Furthermore, as described above, the preparation of the antibody requires the use of an immunized animal, which is not preferable from the viewpoint of animal welfare.
また、抗体は、上述の診断薬などに二次抗体として用いる場合、抗原−抗体複合体への結合の程度を分光光度的に検出するため、ペルオキシダーゼ等の標識化合物とのコンジュゲート体として用いられることが多いが、このようなコンジュゲート体の調製は、二次抗体の調製に加えて、さらに煩雑となる。 In addition, when the antibody is used as a secondary antibody in the above-described diagnostic agents, etc., it is used as a conjugate with a labeling compound such as peroxidase in order to detect the degree of binding to the antigen-antibody complex spectrophotometrically. In many cases, the preparation of such a conjugate is more complicated in addition to the preparation of the secondary antibody.
さらに、現在の抗体市場においては、抗体の由来動物としては、抗体に対応する抗原の由来動物と免疫学的に交叉性を有さない点で、被検動物と異なる動物が挙げられ、例えば、ウサギ、ヤギ、モルモットが含まれる。しかしながら、抗体の由来動物として、マウスも汎用的に用いられており、マウスは、抗体の由来動物として、依然需要の高い動物であると言える。 Furthermore, in the current antibody market, the animal derived from an antibody includes an animal different from the test animal in that it has no immunological crossover with an animal derived from an antigen corresponding to the antibody. Includes rabbits, goats, and guinea pigs. However, mice are also widely used as antibody-derived animals, and it can be said that mice are still in high demand as antibodies-derived animals.
従って、抗体に代わって、抗原と特異的に結合し得る分子種が所望されていた。 Therefore, instead of antibodies, molecular species that can specifically bind to antigens have been desired.
なお、出願人は出願時点までに本発明に関連する公開された先行技術文献を発見することができなかった。よって、先行技術文献情報を開示していない。 The applicant has not been able to find published prior art documents related to the present invention by the time of filing. Therefore, prior art document information is not disclosed.
本発明は、上記の従来の問題に鑑みてなされたものであって、抗体よりも簡便に調製可能で、且つ抗体と比べて同等以上の結合性を有する、マウス由来のＩｇＧ抗体に結合性を有する核酸分子を提供する。 The present invention has been made in view of the above-described conventional problems, and is capable of preparing a mouse-derived IgG antibody that can be more easily prepared than an antibody and has a binding property equal to or higher than that of an antibody. A nucleic acid molecule is provided.
本発明による核酸分子は、マウス由来のＩｇＧ抗体に結合性を有することを特徴とする。 The nucleic acid molecule according to the present invention has a binding property to a mouse-derived IgG antibody.
一方、本発明による検出キットは、マウス由来のＩｇＧ抗体への結合を検出する検出キットであって、上記の核酸分子を含有する試薬を有することを特徴とする。 On the other hand, the detection kit according to the present invention is a detection kit for detecting binding to a mouse-derived IgG antibody, and has a reagent containing the above-mentioned nucleic acid molecule.
本発明によれば、マウス由来のＩｇＧ抗体と特異的に結合し得る核酸分子を得ることができる。 According to the present invention, a nucleic acid molecule capable of specifically binding to a mouse-derived IgG antibody can be obtained.
（本発明による核酸分子）
本発明による核酸分子は、マウス由来のＩｇＧ抗体に結合性を有することを特徴とするものである。
(Nucleic acid molecule according to the present invention)
The nucleic acid molecule according to the present invention is characterized by having a binding property to a mouse-derived IgG antibody.
本発明において、核酸分子とは、アデニン（Ａ）、グアニン（Ｇ）、シトシン（Ｃ）、チミン（Ｔ）、ウラシル（Ｕ）など種々の核酸を有するヌクレオチドであれば、特に制約はなく、ｓｓＤＮＡ、ｓｓＲＮＡ、ｄｓＤＮＡ、ｄｓＲＮＡなど、鎖の本数や、核酸が修飾されているか否か等に制約はない。また、本発明による核酸分子は、マウス由来のＩｇＧ抗体への結合の程度に影響を与えない範囲で、フッ素、塩素、臭素及びヨウ素などのハロゲンや、メチル、エチル、プロピルなどのアルキル基など、適当に置換された核酸分子の置換体も包含する。ＲＮＡに対する修飾核酸としては、そのほかにもＬＮＡ（Ｌｏｃｋｅｄ Ｎｕｃｌｅｉｃ Ａｃｉｄ）、ＥＮＡ（２’−Ｏ，４’−Ｃ−Ｅｔｈｙｌｅｎｅ−ｂｒｉｄｇｅｄ Ｎｕｃｌｅｉｃ Ａｃｉｄｓ）、チオール化などが挙げられる。 In the present invention, the nucleic acid molecule is not particularly limited as long as it is a nucleotide having various nucleic acids such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). , SsRNA, dsDNA, dsRNA, etc. There are no restrictions on the number of strands, whether the nucleic acid is modified, or the like. In addition, the nucleic acid molecule according to the present invention does not affect the degree of binding to mouse-derived IgG antibodies, such as halogens such as fluorine, chlorine, bromine and iodine, and alkyl groups such as methyl, ethyl and propyl, Also included are substitutions of appropriately substituted nucleic acid molecules. Other examples of modified nucleic acids for RNA include LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylene-bridged Nucleic Acids), and thiolation.
本発明において、マウス由来のＩｇＧ抗体のサブクラスとしては、特に制約はなく、例えば、サブクラス１、サブクラス２ａ及びサブクラス３並びにこれらを任意に有する異なるサブクラスを有するＩｇＧ抗体を混合したものが挙げられる。 In the present invention, the subclass of the mouse-derived IgG antibody is not particularly limited, and examples thereof include a mixture of IgG antibodies having subclass 1, subclass 2a and subclass 3, and different subclasses optionally having these.
本発明による核酸分子は、いわゆるＲＮＡプールなどの核酸分子と、標的物質としてのマウス由来のＩｇＧ抗体とを用いて、核酸分子と標的物質とが特異的に結合して形成される核酸分子／標的物質複合体から、この複合体の形成に関与した核酸分子のみを選択する方法で製造することが可能である。このような方法としては、例えば、ＳＥＬＥＸ法（Ｓｙｓｔｅｍａｔｉｃ Ｅｖｏｌｕｔｉｏｎ ｏｆ Ｌｉｇａｎｄｓ ｂｙ Ｅｘｐｏｎｅｎｔｉａｌ Ｅｎｒｉｃｈｍｅｎｔ）と称される方法や、アガロースゲルやポリアクリルアミドゲルなどの担体を用いて核酸分子／標的物質複合体を得た後にこの複合体の形成に関与した核酸分子のみを回収する方法などが挙げられる。 The nucleic acid molecule according to the present invention is a nucleic acid molecule / target formed by specifically binding a nucleic acid molecule and a target substance using a nucleic acid molecule such as a so-called RNA pool and a mouse-derived IgG antibody as a target substance. It is possible to produce a substance complex by a method of selecting only nucleic acid molecules involved in the formation of this complex. Such methods include, for example, a method called SELEX method (Systematic Evolution of Ligands by Exponential Enrichment), or after obtaining a nucleic acid molecule / target substance complex using a carrier such as agarose gel or polyacrylamide gel. Examples include a method of recovering only the nucleic acid molecules involved in the formation of this complex.
（ＳＥＬＥＸ法に準じた本発明による核酸分子の製造方法）
本発明による核酸分子は、ＳＥＬＥＸ法に従って、またこの方法に準じた方法で、ＲＮＡプールと標的物質とを反応させて得られるＲＮＡプール−標的物質複合体を回収した後、この複合体から、この複合体の形成に関与したＲＮＡプールのみを回収して、製造することが可能である。
(Method for producing nucleic acid molecule according to the present invention according to the SELEX method)
The nucleic acid molecule according to the present invention can be obtained by recovering an RNA pool-target substance complex obtained by reacting an RNA pool with a target substance according to the SELEX method or a method according to this method. Only RNA pools involved in complex formation can be recovered and manufactured.
ＲＮＡプールとは、Ａ、Ｇ、Ｃ及びＵからなる群から選択された塩基、及びこの塩基の置換体を２０〜１２０個程度連結した領域（この領域を、以下、「ランダム領域」と称する。）を有する遺伝子配列を総称する遺伝子の混合物をいう。従って、ＲＮＡプールは、４２０〜４１２０（１０１２〜１０７２）種類の複数の遺伝子が含まれ、４３０〜４６０（１０１８〜１０３６）種類の遺伝子が含まれることが好ましい。塩基の置換体としては、フッ素、塩素、臭素及び要素などのハロゲンや、メチル、エチル、プロピルなどのアルキル基など、適当に置換された塩基が挙げられる。また、上記の通り、ＬＮＡ、ＥＮＡ等を用いた修飾、チオール化などされた塩基が挙げられる。 The RNA pool is a region in which about 20 to 120 bases selected from the group consisting of A, G, C, and U and substitutions of this base are linked (this region is hereinafter referred to as “random region”). ) Is a mixture of genes generically referring to gene sequences having. Therefore, the RNA pool preferably includes 4 20 to 4 120 (10 12 to 10 72 ) types of genes, and preferably includes 4 30 to 4 60 (10 18 to 10 36 ) types of genes. Examples of base substitutes include appropriately substituted bases such as fluorine, chlorine, bromine and halogens such as elements, and alkyl groups such as methyl, ethyl and propyl. In addition, as described above, bases modified or thiolated with LNA, ENA or the like can be mentioned.
ＲＮＡプールは、ランダム領域を有する限り、その他の構造に制約はないが、本発明による核酸分子をＳＥＬＥＸ法に準じて製造する場合、ランダム領域の５’末端及び／又は３’末端には、後述のＰＣＲ等で利用するプライマー領域や、ＤＮＡ依存性ＲＮＡポリメラーゼの認識領域を有することが好ましい。例えば、ランダム領域は、５’末端側からＴ７プロモーターなどのＤＮＡ依存性ＲＮＡポリメラーゼの認識領域（以下、この領域を「ＲＮＡポリメラーゼ認識領域」と称する。）と、ＤＮＡ依存性ＤＮＡポリメラーゼのプライマー領域（以下、この領域を「５’末端側プライマー領域」と称する。）とを連結し、この５’末端側プライマー領域の３’末端にランダム領域を連結し、さらにこのランダム領域の３’末端側にＤＮＡ依存性ＤＮＡポリメラーゼのプライマー領域（以下、この領域を、「３’末端側プライマー領域」と称する。）を連結した構造を有してもよい。また、ＲＮＡプールは、これらの領域の他に、標的物質への結合を補助する公知の領域を有してもよい。さらに、ＲＮＡプールは、ランダム領域の一部が各ＲＮＡプールにおいて同じ配列を有するものであってもよい。 The RNA pool is not limited in other structures as long as it has a random region. However, when the nucleic acid molecule according to the present invention is produced according to the SELEX method, the 5 ′ end and / or the 3 ′ end of the random region are described later. It preferably has a primer region used in PCR and the like, and a DNA-dependent RNA polymerase recognition region. For example, the random region includes a DNA-dependent RNA polymerase recognition region such as T7 promoter from the 5 ′ end side (hereinafter, this region is referred to as “RNA polymerase recognition region”) and a DNA-dependent DNA polymerase primer region ( Hereinafter, this region is referred to as “5 ′ terminal primer region”), a random region is connected to the 3 ′ end of this 5 ′ terminal primer region, and further to the 3 ′ end of this random region. It may have a structure in which a primer region of a DNA-dependent DNA polymerase (hereinafter, this region is referred to as “3 ′ terminal primer region”) is linked. In addition to these regions, the RNA pool may have a known region that assists in binding to the target substance. Furthermore, the RNA pool may have a part of the random region having the same sequence in each RNA pool.
ランダム領域は、ＲＮＡプールのランダム領域のＵをＴに置き換えた初期プールを鋳型として、ＰＣＲ法に基づいて、遺伝子増幅した後、得た遺伝子産物と、Ｔ７ポリメラーゼ等のＤＮＡ依存性ＲＮＡポリメラーゼとを反応させて、調製されてもよい。また、初期プールに相補的な遺伝子を合成し、ＲＮＡポリメラーゼ認識領域と、５’末端側プライマー領域に相補的な配列とからなるプライマーを、初期プールにおいてこのプライマーと相補的な遺伝子にアニーリングさせて、ＰＣＲ法に基づいて、調製されてもよい。 The random region is obtained by performing gene amplification based on the PCR method using an initial pool in which U in the random region of the RNA pool is replaced with T as a template, and a DNA-dependent RNA polymerase such as T7 polymerase. It may be prepared by reacting. In addition, a gene complementary to the initial pool is synthesized, and a primer composed of an RNA polymerase recognition region and a sequence complementary to the 5 ′ terminal primer region is annealed to the gene complementary to this primer in the initial pool. It may be prepared based on the PCR method.
次に、このようにして合成したＲＮＡプールと、標的物質であるマウス由来のＩｇＧ抗体とを水素結合などの分子間力を介して結合させる。この結合方法としては、ＲＮＡプールと標的物質とを、標的物質の結合などの機能が保たれる緩衝液中で一定時間インキュベートする方法が挙げられる。このようにして、緩衝液中でＲＮＡプール−標的物質複合体が形成される。 Next, the RNA pool synthesized in this manner and the mouse IgG antibody that is the target substance are bound via intermolecular forces such as hydrogen bonding. Examples of the binding method include a method in which the RNA pool and the target substance are incubated for a certain period of time in a buffer solution that maintains a function such as binding of the target substance. In this way, an RNA pool-target substance complex is formed in the buffer.
次に、このように形成されたＲＮＡプール−標的物質複合体を回収する。緩衝液中には、この複合体の他、複合体の形成に関与しなかったＲＮＡプールや標的物質が含まれるが、この複合体の回収方法としては、標的物質に結合性を有する核酸分子を回収することを目的として、緩衝液中に存在する複合体の形成に関与しなかったＲＮＡプールを除去する方法により行えばよい。この方法としては、標的物質及びＲＮＡプールの特定の成分への吸着性の違いを利用する方法や、複合体とＲＮＡプールとの分子量の違いを利用する方法が挙げられる。 Next, the RNA pool-target substance complex thus formed is recovered. In addition to this complex, the buffer contains RNA pools and target substances that were not involved in the formation of the complex. As a method for recovering this complex, a nucleic acid molecule that binds to the target substance is used. For the purpose of recovery, a method of removing an RNA pool that was not involved in the formation of a complex present in the buffer solution may be used. Examples of this method include a method that utilizes the difference in the adsorptivity of the target substance and the RNA pool to a specific component, and a method that utilizes the difference in molecular weight between the complex and the RNA pool.
標的物質及びＲＮＡプールの特定の成分への吸着性の違いを利用した方法としては、例えば、ニトロセルロース等の標的物質に吸着性を有する膜を用いて、上述のＲＮＡプール−標的物質複合体を有する緩衝液を濾過し、この膜上にＲＮＡプール−標的物質複合体を吸着させ、その後、膜上に残存したＲＮＡプール−標的物質複合体から、複合体の形成に関与したＲＮＡプールを、例えばこの複合体におけるＲＮＡプールと標的物質との結合を解除した後にＲＮＡプールを回収する方法が挙げられる。 As a method using the difference in the adsorptivity to a specific component of the target substance and the RNA pool, for example, the above-mentioned RNA pool-target substance complex can be prepared using a membrane having an adsorptivity to the target substance such as nitrocellulose. The RNA pool-target substance complex is adsorbed on the membrane, and then the RNA pool involved in the formation of the complex from the RNA pool-target substance complex remaining on the membrane is, for example, A method of recovering the RNA pool after releasing the binding between the RNA pool and the target substance in this complex can be mentioned.
また、ＲＮＡプール−標的物質複合体とＲＮＡプールとの分子量の違いを利用した方法としては、アガロースゲルなど、ＲＮＡプールを通過させ得るがＲＮＡプール−標的物質複合体を通過させ得ない程度のポアを有する担体を利用して、ＲＮＡプール−標的物質複合体とＲＮＡプールとを電気的に分離し、この複合体から、複合体の形成に関与したＲＮＡプールを回収する方法が挙げられる。 Further, as a method using the difference in molecular weight between the RNA pool-target substance complex and the RNA pool, a pore such as an agarose gel that can pass through the RNA pool but cannot pass through the RNA pool-target substance complex. There is a method in which an RNA pool-target substance complex and an RNA pool are electrically separated using a carrier having the following, and an RNA pool involved in the formation of the complex is recovered from this complex.
次に、このようにして得たＲＮＡプール−標的物質複合体から回収した複合体の形成に関与したＲＮＡプールを用いて、遺伝子増幅を行う。この遺伝子増幅の方法としては、ＲＮＡプールに含まれる５’末端側プライマー領域、３’末端側プライマー領域、ＲＮＡポリメラーゼ認識領域を利用する方法が挙げられる。例えば、複合体の形成に関与したＲＮＡプールの３’末端側プライマー領域に相補的な遺伝子断片をプライマーとして用いて、トリ骨髄芽球症ウィルス由来リバーストランスクリプターゼ（ＡＭＶ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ）などのＲＮＡ依存性ＤＮＡポリメラーゼを用いた逆転写反応に従ってｃＤＮＡを調製した後、このｃＤＮＡに含まれる５’末端側プライマー領域及び３’末端側プライマー領域を利用して、ＤＮＡ依存性ＤＮＡポリメラーゼを用いたＰＣＲ反応を行い、得た遺伝子産物に含まれるＲＮＡポリメラーゼ認識領域を利用して、ＤＮＡ依存性ＲＮＡポリメラーゼを用いて、ｉｎ ｖｉｔｒｏ転写反応を行って、ＲＮＡプールの遺伝子増幅を行ってもよい。 Next, gene amplification is performed using the RNA pool involved in the formation of the complex recovered from the RNA pool-target substance complex thus obtained. Examples of the gene amplification method include a method using a 5'-end primer region, a 3'-end primer region, and an RNA polymerase recognition region contained in the RNA pool. For example, RNA dependency such as avian myeloblastosis virus-derived reverse transcriptase (AMV Reverse Transscriptase) using a gene fragment complementary to the 3 ′ terminal primer region of the RNA pool involved in complex formation as a primer After preparing a cDNA according to a reverse transcription reaction using a sexual DNA polymerase, a PCR reaction using a DNA-dependent DNA polymerase is carried out using the 5 ′ terminal primer region and the 3 ′ terminal primer region contained in this cDNA. The RNA pool may be amplified by performing an in vitro transcription reaction using a DNA-dependent RNA polymerase using an RNA polymerase recognition region contained in the obtained gene product.
このように遺伝子増幅された複合体の形成に関与したＲＮＡプールと、標的物質とを用いて、上述のＲＮＡプール−標的物質複合体を形成する方法以下の各方法を繰り返し行い、最終的に、標的物質としてのマウス由来のＩｇＧ抗体に特異的に結合する核酸分子、つまり、マウス由来のＩｇＧ抗体に結合性を有する核酸分子を得ることができる。 Using the RNA pool involved in the formation of the complex thus amplified and the target substance, the method of forming the RNA pool-target substance complex described above is repeated, and finally, A nucleic acid molecule that specifically binds to a mouse-derived IgG antibody as a target substance, that is, a nucleic acid molecule that binds to a mouse-derived IgG antibody can be obtained.
（本発明における二次構造予測）
本発明による核酸分子は、上記の通りに得ることが出来るが、その塩基配列に基づいた二次構造予測の手法を用いた結果を参照して、得た核酸分子の一部を改変されたものであってもよい。この二次構造予測としては、核酸分子の二次構造の候補を探索し、この探索された二次構造候補のうち、エネルギー的に安定な二次構造を予測する方法であれば、特に制約はない。例えば、核酸分子の塩基配列を、ワトソン・クリック型などの塩基対を構成するステム領域と、このステム領域以外の塩基で構成されるループ構造などの一本鎖領域とに分割して得た二次構造候補のエネルギー関数を最小化することに基づく二次構造予測であってもよい。
(Secondary structure prediction in the present invention)
The nucleic acid molecule according to the present invention can be obtained as described above, but a part of the obtained nucleic acid molecule is modified with reference to the result of using the secondary structure prediction method based on the base sequence. It may be. The secondary structure prediction is particularly limited as long as it is a method for searching for secondary structure candidates of nucleic acid molecules and predicting secondary structures that are energetically stable among the searched secondary structure candidates. Absent. For example, two nucleic acid molecules obtained by dividing the base sequence of a nucleic acid molecule into a stem region comprising a base pair such as Watson-Crick type and a single-stranded region such as a loop structure comprising a base other than this stem region Secondary structure prediction based on minimizing the energy function of the secondary structure candidate may be used.
この二次構造候補のエネルギー関数を最小化することに基づく二次構造予測について、説明する。まず、対象となる核酸分子の塩基配列のうち、ワトソン・クリック型などの塩基対を構成する塩基の候補と、この塩基対候補以外の一本鎖領域の候補とを探索する。探索された塩基対候補及び一本鎖領域候補の全組合せのうち、塩基対候補を構成する塩基と一本鎖領域候補を構成する塩基とが重複するなど、理論的に取り得ない組合せを除いて、二次構造候補を同定する。同定された二次構造候補のうち、二次構造候補のエネルギー関数を算出し、算出されたエネルギー関数が最小となる二次構造を探索する。この際、この二次構造候補のエネルギー関数の算出方法としては、二次構造候補を構成する個々のステム領域及び一本鎖領域の自由エネルギーに基づいて、この二次構造候補における自由エネルギーを、二次構造候補のエネルギー関数とする方法であってもよい。このようにして、同定された二次構造候補のうち、エネルギー関数の最も小さい二次構造を、対象となる核酸分子の塩基配列の二次構造とする。 The secondary structure prediction based on minimizing the energy function of the secondary structure candidate will be described. First, in the base sequence of the target nucleic acid molecule, a candidate for a base constituting a Watson-Crick base pair and a single-stranded region candidate other than this base pair candidate are searched. Except for combinations that cannot be theoretically taken, such as the bases that make up the base pair candidates and the bases that make up the single-stranded region candidates overlap among all the combinations of searched base pair candidates and single-stranded region candidates Identify secondary structure candidates. Among the identified secondary structure candidates, the energy function of the secondary structure candidate is calculated, and the secondary structure that minimizes the calculated energy function is searched. At this time, as a method of calculating the energy function of this secondary structure candidate, based on the free energy of each stem region and single-stranded region constituting the secondary structure candidate, the free energy in this secondary structure candidate is A method of using an energy function of a secondary structure candidate may be used. Thus, among the identified secondary structure candidates, the secondary structure having the smallest energy function is set as the secondary structure of the base sequence of the target nucleic acid molecule.
本発明による核酸分子は、このようにして得た二次構造の結果を参照して、二次構造のうちの特徴ある部位を構成する塩基の置換若しくは欠失によって、又は二次構造のうちの特徴ある部分への塩基の挿入などによって、改変されてもよい。例えば、上記の通り調製した核酸分子を親分子として、二次構造のステム領域及び／又は一本鎖領域を構成する塩基の一部を置換してもよい。また、二次構造のステム領域及び／又は一本鎖領域を構成する塩基の一部を欠失させてもよい。また、二次構造のステム領域及び／又は一本鎖領域に、単数又は複数の塩基を挿入して、ステム長さ及び／又は一本鎖領域の長さを短縮／延長してもよい。 The nucleic acid molecule according to the present invention refers to the result of the secondary structure thus obtained, by substitution or deletion of the base constituting the characteristic site of the secondary structure, or of the secondary structure. It may be modified by insertion of a base into a characteristic part. For example, a part of the base constituting the stem region and / or the single-stranded region of the secondary structure may be substituted with the nucleic acid molecule prepared as described above as the parent molecule. Further, a part of the base constituting the stem region and / or the single-stranded region of the secondary structure may be deleted. Alternatively, the stem length and / or the length of the single-stranded region may be shortened / extended by inserting one or more bases into the stem region and / or single-stranded region of the secondary structure.
本発明による核酸分子は、この二次構造予測を用いて得た核酸分子の二次構造において、ステム長さ３以上のステム領域を、この核酸分子の末端に有することが好ましい。また、本発明による核酸分子において、ステム長さ３以上のステム領域の一本鎖領域側の末端の塩基に隣接する塩基が、アデニン以外の塩基であることが好ましい。また、本発明による核酸分子において、ステム長さ３以上のステム領域は、グアニン残基及びシトシン残基のみから構成されていることが好ましい。これらにより、マウス由来のＩｇＧ抗体への結合性がさらに向上される。 The nucleic acid molecule according to the present invention preferably has a stem region having a stem length of 3 or more at the end of the nucleic acid molecule in the secondary structure of the nucleic acid molecule obtained by using this secondary structure prediction. In the nucleic acid molecule according to the present invention, the base adjacent to the terminal base on the single-stranded region side of the stem region having a stem length of 3 or more is preferably a base other than adenine. In the nucleic acid molecule according to the present invention, the stem region having a stem length of 3 or more is preferably composed of only guanine residues and cytosine residues. These further improve the binding properties to mouse-derived IgG antibodies.
（本発明による核酸分子の用途等）
本発明による核酸分子は、上記の通り、マウス由来のＩｇＧ抗体に結合性を有することを特徴とするものである。従って、本発明による核酸分子の用途としては、マウス由来のＩｇＧ抗体への結合性を利用する用途であれば特に制約はなく、ＳＤＳ−ＰＡＧＥ（ＳＤＳポリアクリルアミド電気泳動法）法に準じて行われる泳動対象物の検出用として、ＥＬＩＳＡ法（酵素免疫測定法；ｅｎｚｙｍｅ−ｌｉｎｋｅｄ ｉｍｍｕｎｏｓｏｒｂｅｎｔ ａｓｓａｙ）の測定の対象物若しくはこの対象物からなる複合体の検出用として、ノースウェスタン法に準じて行われる泳動対象物の検出用として、又はマウス由来のＩｇＧ抗体若しくはこの抗体を利用した精製用として用いられてもよい。
(Use of nucleic acid molecule according to the present invention)
As described above, the nucleic acid molecule according to the present invention has a binding property to a mouse-derived IgG antibody. Therefore, the use of the nucleic acid molecule according to the present invention is not particularly limited as long as it uses the binding property to a mouse-derived IgG antibody, and is performed according to the SDS-PAGE (SDS polyacrylamide electrophoresis) method. For the detection of the electrophoresis target, the target for the measurement of the ELISA method (enzyme-linked immunosorbent assay) or the complex consisting of the target is performed according to the Northwestern method. It may be used for detection of an object, or for purification using a mouse-derived IgG antibody or this antibody.
例えば、本発明による核酸分子をＳＤＳ−ＰＡＧＥ法に用いる場合には、泳動されたタンパク質を検出するために用いられてもよく、泳動されたタンパク質を検出するのに用いられたマウス由来のＩｇＧ抗体を検出するのに用いられてもよい。 For example, when the nucleic acid molecule according to the present invention is used in the SDS-PAGE method, it may be used to detect the migrated protein, and the mouse-derived IgG antibody used to detect the migrated protein May be used to detect.
また、本発明による核酸分子をＥＬＩＳＡ法に用いる場合には、測定の対象となるマウスＩｇＧを検出するのに用いられてもよく、また、測定の対象を検出するために用いられたマウス由来のＩｇＧ抗体を検出するのに用いられてもよい。 When the nucleic acid molecule according to the present invention is used in the ELISA method, it may be used to detect mouse IgG to be measured, or derived from the mouse used to detect the measurement target. It may be used to detect IgG antibodies.
また、本発明による核酸分子をノースウェスタン法に用いる場合には、電気泳動の対象となるマウス由来のＩｇＧ抗体を検出するのに用いられてもよく、また、泳動の対象となるタンパク質の検出に用いられるマウス由来のＩｇＧ抗体を検出するのに用いられてもよい。 In addition, when the nucleic acid molecule according to the present invention is used for the Northwestern method, it may be used for detecting an IgG antibody derived from a mouse to be electrophoresed, or for detecting a protein to be electrophoresed. It may be used to detect the mouse-derived IgG antibody used.
また、本発明による核酸分子をマウス由来のＩｇＧの精製に用いる場合、精製の対象となるマウス由来のＩｇＧ抗体を精製するのに適当な形態で利用すればよく、例えば、本発明による核酸分子を、アガロースや合成樹脂からなるビーズに結合して、使用してもよい。このようにビーズに結合する形態としては、精製に用いる担体（例えば、アガロースや、合成樹脂からなるビーズ）に結合性を有するように、ビオチン化など、種々の改変が挙げられる。従って、本発明による核酸分子は、ビオチン化などの種々の改変を受けたものであってもよい。 Further, when the nucleic acid molecule according to the present invention is used for purifying mouse-derived IgG, it may be used in a form suitable for purifying the mouse-derived IgG antibody to be purified. For example, the nucleic acid molecule according to the present invention is used. Alternatively, it may be used by binding to beads made of agarose or synthetic resin. Thus, the form couple | bonded with a bead includes various modification | changes, such as biotinylation, so that it may have a binding property to the support | carrier (For example, bead which consists of agarose and a synthetic resin) used for refinement | purification. Therefore, the nucleic acid molecule according to the present invention may have undergone various modifications such as biotinylation.
（本発明による核酸分子の応用例）
本発明による核酸分子の応用例としては、得た核酸分子を試薬、この試薬を有するキットなど、マウス由来のＩｇＧ抗体への結合性を利用したものが挙げられる。例えば、上記の本発明による核酸分子を含有する試薬を用いて、マウス由来のＩｇＧ抗体への結合を検出する検出キットとしてもよい。このようなキットの測定対象物としては、溶液、懸濁液のような液体系であってもよく、培養細胞や組織切片など、固形物であってもよい。
(Application example of nucleic acid molecule according to the present invention)
Examples of the application of the nucleic acid molecule according to the present invention include those using the obtained nucleic acid molecule as a reagent, a kit having this reagent, and the like, which utilizes binding properties to mouse-derived IgG antibodies. For example, a detection kit for detecting binding to a mouse-derived IgG antibody using the above-described reagent containing the nucleic acid molecule according to the present invention may be used. The measurement object of such a kit may be a liquid system such as a solution or a suspension, or may be a solid material such as a cultured cell or tissue section.
また、本発明による検出キットにおいて、本発明による核酸分子以外の試薬としては、標的物質と核酸分子との結合を検出するために必要な物質を適宜選択すればよく、これは、検出キットに用いる核酸分子によって適宜選択すればよい。 In the detection kit according to the present invention, as a reagent other than the nucleic acid molecule according to the present invention, a substance necessary for detecting the binding between the target substance and the nucleic acid molecule may be appropriately selected, and this is used for the detection kit. What is necessary is just to select suitably with a nucleic acid molecule.
例えば、本発明による検出キットは、ＳＤＳ−ＰＡＧＥ法（ＳＤＳポリアクリルアミド電気泳動法）に準じて行うものであってもよく、この場合、検出キットに含まれる試薬としては、電気泳動の対象となるタンパク質の検出に用いるマウスＩｇＧに結合性を有する核酸分子であってもよく、また電気泳動の対象となるマウスＩｇＧに結合性を有する核酸分子であってもよい。 For example, the detection kit according to the present invention may be performed in accordance with the SDS-PAGE method (SDS polyacrylamide electrophoresis), and in this case, the reagent contained in the detection kit is subject to electrophoresis. It may be a nucleic acid molecule having binding property to mouse IgG used for protein detection, or may be a nucleic acid molecule having binding property to mouse IgG to be subjected to electrophoresis.
また、本発明による検出キットは、ＥＬＩＳＡ法（酵素免疫測定法；ｅｎｚｙｍｅ−ｌｉｎｋｅｄ ｉｍｍｕｎｏｓｏｒｂｅｎｔ ａｓｓａｙ）に準じて行うものであってもよく、この場合、検出キットに含まれる試薬としては、測定の対象となるマウスＩｇＧに結合性を有する核酸分子が挙げられる。 In addition, the detection kit according to the present invention may be performed in accordance with an ELISA method (enzyme-linked immunosorbent assay). In this case, the reagents contained in the detection kit include the target of measurement. And a nucleic acid molecule having binding property to mouse IgG.
また、本発明による検出キットは、ノースウェスタン法に準じて行われるものであってもよく、この場合、検出キットに含まれる試薬としては、泳動の対象となるタンパク質の検出に用いられるマウスＩｇＧに結合性を有する核酸分子であってもよく、また電気泳動の対象となるマウスＩｇＧに結合性を有する核酸分子であってもよい。 In addition, the detection kit according to the present invention may be performed according to the Northwestern method. In this case, as a reagent contained in the detection kit, mouse IgG used for detection of a protein to be electrophoresed is used. It may be a nucleic acid molecule having a binding property or a nucleic acid molecule having a binding property to mouse IgG to be subjected to electrophoresis.
（製造例１）
配列番号１３に示す初期プールを、北海道システム・サイエンス株式会社に依頼して合成した。この初期プール（５００ｎＭ）と、プライマー１（配列番号１４）と、プライマー２（配列番号１５）と、５Ｕ／μＬのＤＮＡポリメラーゼ（商品名：ＴａＫａＲａ Ｅｘ−Ｔａｑ、タカラバイオ株式会社製、カタログ番号：ＲＲ００１Ａ）とを用いて、初期プールと、初期プールに相補的な遺伝子鎖とからなるｃＤＮＡを得た。次に、このようにして得たｃＤＮＡと、ＡｍｐｌｉＳｃｒｉｂｅ（登録商標）Ｔ７ Ｈｉｇｈ Ｙｉｅｌｄ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｋｉｔ（ＥＰＩＣＥＮＴＥＲ社製）とを用いて、転写反応を行い、ＲＮＡプール（配列番号１６）を得た。
(Production Example 1)
The initial pool shown in SEQ ID NO: 13 was synthesized by requesting Hokkaido System Science Co., Ltd. This initial pool (500 nM), primer 1 (SEQ ID NO: 14), primer 2 (SEQ ID NO: 15), 5 U / μL DNA polymerase (trade name: TaKaRa Ex-Taq, manufactured by Takara Bio Inc., catalog number: RR001A) was used to obtain a cDNA comprising an initial pool and a gene chain complementary to the initial pool. Next, a transcription reaction was performed using the cDNA thus obtained and Ampliscribe (registered trademark) T7 High Yield Transfer Kit (manufactured by EPICENTER) to obtain an RNA pool (SEQ ID NO: 16).
このようにして得たＲＮＡプールと、下記の標的物質１〜４とを、結合バッファー（２０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ７．４、１５０ｍＭ ＮａＣｌ、５ｍＭ ＭｇＣｌ２）中で、室温、２０分間インキュベートした。 The RNA pool thus obtained and the following target substances 1 to 4 were incubated in a binding buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl 2 ) at room temperature for 20 minutes.
標的物質１：マウスＩｇＧ抗体（サンタクルス社製、カタログ番号ｓｃ−２０２５、０．１％アジ化ナトリウム及び０．１％ゼラチンを含有）であって、ゼラチンを取り除くためにカラム（Ｐｒｏ−ｃｈｅｍ社製 ＰＲＯＴＥＵＳ Ｐｒｏｔｅｉｎ Ｇ Ｍｉｎｉ Ｋｉｔ カタログ番号ＰＣ−ＭＧＫ１６）で精製したもの
標的物質２：マウス由来抗ＦＬＡＧ−ＩｇＧ抗体（ＳＩＧＭＡ社製、カタログ番号Ｆ３１６５、サブタイプ１）
標的物質３：マウス由来抗ＭＢＰ−ＩｇＧ抗体（ＮＥＢ社製、カタログ番号Ｅ８０３２Ｓ、サブタイプ２ａ）
標的物質４：マウス由来抗体ＭＢＰ−ＩｇＧ抗体（ＭＢＬ社製、カタログ番号Ｍ０９１−３、サブタイプ３）
Target substance 1: mouse IgG antibody (manufactured by Santa Cruz, catalog number sc-2025, containing 0.1% sodium azide and 0.1% gelatin), and a column (Pro-chem) for removing gelatin Purified by PROTEUS Protein G Mini Kit, catalog number PC-MGK16) Target substance 2: Mouse-derived anti-FLAG-IgG antibody (manufactured by SIGMA, catalog number F3165, subtype 1)
Target substance 3: mouse-derived anti-MBP-IgG antibody (manufactured by NEB, catalog number E8032S, subtype 2a)
Target substance 4: Mouse-derived antibody MBP-IgG antibody (manufactured by MBL, catalog number M091-3, subtype 3)
その後、得た混合物について、下記の[ゲル法］に準じて、第一のサイクルを行い、その後、下記の［フィルター法］に準じて、第二から第七のサイクルを行った。 Thereafter, the obtained mixture was subjected to the first cycle according to the following [Gel method], and then the second to seventh cycles were performed according to the following [Filter method].
このようにして配列番号１〜４に示すＲＮＡを得た。 In this way, RNAs shown in SEQ ID NOs: 1 to 4 were obtained.
［ゲル法］
得た混合物を、１％のアガロースゲルのウェルに導入し、ＴＢ緩衝液（４４．５ｍＭ Ｔｒｉｓ、４４．５ｍＭ ホウ酸、５ｍＭ ＭｇＣｌ２、ｐＨ８．０）中、１００Ｖで７分間、電気泳動を行った。その後、ウェルに残存する液を回収した。
[Gel method]
The obtained mixture was introduced into a well of a 1% agarose gel and subjected to electrophoresis at 100 V for 7 minutes in TB buffer (44.5 mM Tris, 44.5 mM boric acid, 5 mM MgCl 2 , pH 8.0). It was. Thereafter, the liquid remaining in the well was collected.
その後、回収した溶液は、エタノール沈殿を行った。その後、プライマー３（配列番号１７）と、ＡＭＶ由来逆転写酵素トランスクリプターゼ（ロシュ社製）とを用いて、５５℃で、３０分間、及び８５℃で５分間、逆転写反応を行った。 Thereafter, the collected solution was subjected to ethanol precipitation. Thereafter, a reverse transcription reaction was performed at 55 ° C. for 30 minutes and at 85 ° C. for 5 minutes using Primer 3 (SEQ ID NO: 17) and AMV-derived reverse transcriptase transcriptase (Roche).
この反応産物全量と、５ＵのＤＮＡポリメラーゼ（商品名：Ｅｘ−Ｔａｑ、タカラバイオ株式会社製）と、３０ｎＭのプライマー１（配列番号１４）と、プライマー２（配列番号１５）とを用いて、９０℃×５０秒、５３℃×７０秒及び７４℃×５０秒の順で行うサイクルを１サイクルとして、３サイクルでＰＣＲ反応を行った。得た液にエタノール沈殿を施し、二本鎖ＤＮＡ産物を得た。 Using the total amount of this reaction product, 5 U DNA polymerase (trade name: Ex-Taq, manufactured by Takara Bio Inc.), 30 nM primer 1 (SEQ ID NO: 14), and primer 2 (SEQ ID NO: 15), 90 The PCR reaction was carried out in 3 cycles, with one cycle consisting of 50 ° C. × 50 seconds, 53 ° C. × 70 seconds, and 74 ° C. × 50 seconds. The obtained solution was subjected to ethanol precipitation to obtain a double-stranded DNA product.
この二本鎖ＤＮＡ産物を８μＬのＲＮａｓｅフリー水に溶解し、そのうち４μＬと、１６μＬのＴ７ＲＮＡポリメラーゼ溶液（商品名：Ａｍｐｌｉｓｃｒｉｂｅ（ＥＰＩＣＥＮＴＲＥ社製））とを用いて、ｉｎ ｖｉｔｒｏ転写を行い、ｉｎ ｖｉｔｒｏ転写物を得た。なお、ここまでの工程を１サイクルと定義する。 This double-stranded DNA product is dissolved in 8 μL of RNase-free water, and in vitro transcription is performed using 4 μL of the product and 16 μL of T7 RNA polymerase solution (trade name: Ampliscribe (manufactured by EPICENTRE)). I got a thing. In addition, the process so far is defined as one cycle.
［フィルター法］
得た混合物を、ポップトップホルダーに固定したニトロセルロース膜に導入してろ過し、膜を１ｍＬの結合バッファーで洗浄した。その後、この膜を４００μＬの溶出バッファー（２０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ７．４、１５０ｍＭ ＮａＣｌ、７Ｍ 尿素）に浸漬して、９０℃、５分間加温した。得た液にエタノール沈殿を施し、オリゴヌクレオチドを得た。
[Filter method]
The obtained mixture was introduced into a nitrocellulose membrane fixed to a poptop holder and filtered, and the membrane was washed with 1 mL of a binding buffer. Thereafter, this membrane was immersed in 400 μL of an elution buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 7 M urea) and heated at 90 ° C. for 5 minutes. The obtained solution was subjected to ethanol precipitation to obtain an oligonucleotide.
その後、このオリゴヌクレオチド全量と、プライマー３（配列番号１７）と、ＡＭＶ由来逆転写酵素トランスクリプターゼ（１０Ｕ、ロシュ・ダイアグノスティックス社製）とを用いて、５５℃で、３０分間、及び８５℃で５分間、逆転写反応を行った。 Thereafter, using this whole amount of oligonucleotide, primer 3 (SEQ ID NO: 17), and AMV-derived reverse transcriptase transcriptase (10 U, manufactured by Roche Diagnostics) at 55 ° C. for 30 minutes, and The reverse transcription reaction was performed at 85 ° C. for 5 minutes.
この反応産物全量と、５ＵのＤＮＡポリメラーゼ（商品名：Ｅｘ−Ｔａｑ、タカラバイオ株式会社製）と、３０ｎＭのプライマー１（配列番号１４）と、プライマー２（配列番号１５）とを用いて、９０℃×５０秒、５３℃×７０秒及び７４℃×５０秒の順で行うサイクルを１サイクルとして、４サイクル以上でＰＣＲ反応を行った。得た液にエタノール沈殿を施し、二本鎖ＤＮＡ産物を得た。 Using the total amount of this reaction product, 5 U DNA polymerase (trade name: Ex-Taq, manufactured by Takara Bio Inc.), 30 nM primer 1 (SEQ ID NO: 14), and primer 2 (SEQ ID NO: 15), 90 The PCR reaction was carried out in 4 cycles or more, with one cycle consisting of 50 ° C. × 50 seconds, 53 ° C. × 70 seconds, and 74 ° C. × 50 seconds. The obtained solution was subjected to ethanol precipitation to obtain a double-stranded DNA product.
この二本鎖ＤＮＡ産物を８μＬのＲＮａｓｅフリー水に溶解し、そのうち４μＬと、２μＬのＴ７ＲＮＡポリメラーゼ（商品名：Ａｍｐｌｉｓｃｒｉｂｅ（ＥＰＩＣＥＮＴＲＥ社製））とを用いて、ｉｎ ｖｉｔｒｏ転写を行い、ｉｎ ｖｉｔｒｏ転写物を得た。なお、ここまでの工程を１サイクルと称する。 This double-stranded DNA product is dissolved in 8 μL of RNase-free water, 4 μL of which is used, and 2 μL of T7 RNA polymerase (trade name: Ampliscribe (manufactured by EPICENTRE)) is used for in vitro transcription, in vitro transcript Got. In addition, the process so far is called 1 cycle.
（実施例１）
配列番号１〜４について、表面プラズモン共鳴（Ｓｕｒｆａｃｅ Ｐｌａｓｍｏｎ Ｒｅｓｏｎａｎｃｅ）を利用したバイオセンサーＢｉａｃｏｒｅ３０００（ビアコア社製）を用いて、以下の通り測定した。
Example 1
About sequence number 1-4, it measured as follows using the biosensor Biacore3000 (made by Biacore) using surface plasmon resonance (Surface Plasmon Resonance).
まず、リガンドとして、配列番号１〜４に示す各ＲＮＡの３’末端側にアデニン２４塩基を連結したものを、定法に従い調製した。５’末端側にビオチンを標識した２４塩基のデオキシチミンをセンサーチップＳＡ（ビアコア社製）に固定化し、つぎに、調製したＲＮＡをデオキシチミンと結合させた。これに、２００ｎＭの標的物質５〜８を、流速２０μＬ／分で２分間添加し、測定物の結合を観察した後、３分間にわたって解離を観察した。 First, a ligand in which 24 bases of adenine was linked to the 3 'end of each RNA shown in SEQ ID NOs: 1 to 4 was prepared according to a conventional method. 24-base deoxythymine labeled with biotin on the 5 'end side was immobilized on a sensor chip SA (manufactured by Biacore), and then the prepared RNA was bound to deoxythymine. To this, 200 nM of the target substance 5-8 was added at a flow rate of 20 μL / min for 2 minutes, and after observing the binding of the analyte, dissociation was observed for 3 minutes.
標的物質５：マウスＩｇＧ抗体（Ｎｏｒｍａｌ ｍｏｕｓｅＩｇＧ（SantaCruz社製）、カタログ番号：ｓｃ−２０２５、サブタイプ：混合）
標的物質６：マウスＩｇＧ抗体（商品名、ａｎｔｉ−ＦＬＡＧ Ｍ２、シグマ社製、カタログ番号：Ｆ３１６５、サブタイプ：１）
標的物質７：マウスＩｇＧ抗体（商品名Ｐｒｏｔｅｉｎ Ａ Ｐｕｒｉｆｉｅｄ Ｍｏｕｓｅ Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉ−ＧＳＴ（Ｒｏｃｋｌａｎｄ社製）、カタログ番号：２００−３０１−２００、サブタイプ：１）
標的物質８：マウスＩｇＧ抗体（ｍｏｕｓｅ ＩｇＧ Ｆｃ ｆｒａｇｍｅｎｔ（Ｒｏｃｋｌａｎｄ社製）、カタログ番号：０１０−０１０３、サブタイプ：混合）
Target substance 5: Mouse IgG antibody (Normal mouse IgG (SantaCruz), catalog number: sc-2025, subtype: mixed)
Target substance 6: Mouse IgG antibody (trade name, anti-FLAG M2, manufactured by Sigma, catalog number: F3165, subtype: 1)
Target substance 7: Mouse IgG antibody (trade name: Protein A Purified Mouse Monoclonal Anti-GST (manufactured by Rockland), catalog number: 200-301-200, subtype: 1)
Target substance 8: Mouse IgG antibody (mouse IgG Fc fragment (manufactured by Rockland), catalog number: 010-0103, subtype: mixed)
反応は２５℃で行った。この観察で結合の有無を観察した。結果を表１に示す。表１中、「＋」印は、結合の見られたものを示しており、コントロールと比較してノイズ幅よりも大きなシグナルが観察されたものに相当する。 The reaction was performed at 25 ° C. With this observation, the presence or absence of binding was observed. The results are shown in Table 1. In Table 1, the “+” mark indicates that binding was observed, and corresponds to the signal in which a signal larger than the noise width was observed as compared with the control.
また、上記の２００ｎＭの標的物質５〜８に代えて、２００ｎＭ、１００ｎＭ、５０ｎＭ、２５ｎＭ、１２．５ｎＭ及び６．２５ｎＭのマウスＩｇＧ抗体（商品名、ａｎｔｉ−ＦＬＡＧ Ｍ２、シグマ社製、カタログ番号：Ｆ３１６５、サブタイプ：１）を用いて得たセンサグラムから、各配列の結合定数（Ｋｄ値）を算出した。また、これらの結合定数の値について、χ（カイ）２乗検定を行った。結果を表２に示す。 Further, instead of the above 200 nM target substances 5-8, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM mouse IgG antibodies (trade name, anti-FLAG M2, manufactured by Sigma, catalog number: The binding constant (Kd value) of each sequence was calculated from the sensorgram obtained using F3165, subtype: 1). In addition, χ (chi) square test was performed on the values of these binding constants. The results are shown in Table 2.
（製造例２）
配列番号１に記載のＲＮＡについて、上記の二次構造予測を行った。具体的には、対象となる配列番号１に示す塩基配列において、ワトソン・クリック型などの塩基対を構成する塩基の候補と、この塩基対候補以外の一本鎖領域の候補とを探索した。探索された塩基対候補及び一本鎖領域候補の全組合せのうち、塩基対候補を構成する塩基と一本鎖領域候補を構成する塩基とが重複するなど、理論的に取り得ない組合せを除いて、二次構造候補を同定した。同定された二次構造候補のうち、二次構造候補のエネルギー関数を算出し、算出されたエネルギー関数が最小となる二次構造を探索した。この際、この二次構造候補のエネルギー関数の算出方法としては、二次構造候補を構成する個々のステム領域及び一本鎖領域の自由エネルギーに基づいて、この二次構造候補における自由エネルギーを、二次構造候補のエネルギー関数とした。このようにして、同定された二次構造候補のうち、エネルギー関数の最も小さい二次構造を、対象となる核酸分子の塩基配列の二次構造とした。その結果を図１に示す。
(Production Example 2)
The above secondary structure prediction was performed for the RNA described in SEQ ID NO: 1. Specifically, in the target base sequence shown in SEQ ID NO: 1, a candidate for a base constituting a Watson-Crick type base pair and a single-stranded region candidate other than this base pair candidate were searched. Except for combinations that cannot be theoretically taken, such as the bases that make up the base pair candidates and the bases that make up the single-stranded region candidates overlap among all the combinations of searched base pair candidates and single-stranded region candidates Secondary structure candidates were identified. Among the identified secondary structure candidates, the energy function of the secondary structure candidate was calculated, and the secondary structure that minimizes the calculated energy function was searched. At this time, as a method of calculating the energy function of this secondary structure candidate, based on the free energy of each stem region and single-stranded region constituting the secondary structure candidate, the free energy in this secondary structure candidate is The energy function of the secondary structure candidate was used. In this way, among the identified secondary structure candidates, the secondary structure having the smallest energy function is set as the secondary structure of the base sequence of the target nucleic acid molecule. The result is shown in FIG.
このようにして得た配列番号１の二次構造の結果を参照すると、１６〜１９残基目及び４５〜４８残基目の塩基が４塩基対からなるステム長を有するステム領域を形成することが分かった。そこで、このステム領域の末端側のいわゆるプライマー領域を欠失させ、グアノシン及びシチジンを用いて、ステム領域のステム長さを２つ延ばし、５’末端側にＧを付加した配列番号５に示すＲＮＡを合成した。また、同様に、ステム領域のステム長さを２つ延ばすなどした配列番号６に示すＲＮＡを合成した。さらに、配列番号６のステム領域に対応する配列を、グアニン残基及びシトシン残基のみからなるものとした配列番号７に示すＲＮＡを合成した。 Referring to the result of the secondary structure of SEQ ID NO: 1 thus obtained, the bases at residues 16-19 and 45-48 form a stem region having a stem length consisting of 4 base pairs. I understood. Therefore, the RNA shown in SEQ ID NO: 5 is obtained by deleting the so-called primer region on the terminal side of the stem region, extending the stem length of the stem region by two using guanosine and cytidine, and adding G to the 5 ′ terminal side. Was synthesized. Similarly, the RNA shown in SEQ ID NO: 6 was synthesized by extending the stem length of the stem region by two. Furthermore, RNA shown in SEQ ID NO: 7 was synthesized in which the sequence corresponding to the stem region of SEQ ID NO: 6 consisted of only a guanine residue and a cytosine residue.
（実施例２）
実施例１において、配列番号１〜４の代わりに、配列番号５〜７を用い、標的物質５〜８の代わりに、下記の標的物質９〜１２の各標的物質を用いた以外は、実施例１と同様に行い、結合の有無を観察した。その結果を表３に示す。
(Example 2)
In Example 1, except that SEQ ID NOs: 5 to 7 were used instead of SEQ ID NOs: 1 to 4, and each target substance of the following target substances 9 to 12 was used instead of the target substances 5 to 8, Example 1 and the presence or absence of binding was observed. The results are shown in Table 3.
標的物質９：マウスＩｇＧ抗体（mouse IgG Isotype Control IgG１（BD Biosciences社製）、カタログ番号：５５７２７３、サブタイプ：１）
標的物質１０：マウスＩｇＧ抗体（mouse IgG Isotype Control IgG２ａ（BD Biosciences社製）、カタログ番号：５５３４５４、サブタイプ：２ａ）
標的物質１１：マウスＩｇＧ抗体（mouse IgG Isotype Control IgG２ｂ（BD Biosciences社製）、カタログ番号：５５７３５１、サブタイプ：２ｂ）
標的物質１２：マウスＩｇＧ抗体（mouse IgG Isotype Control IgG３（BD Biosciences社製）、カタログ番号：５５３４８６、サブタイプ：３）
Target substance 9: Mouse IgG antibody (mouse IgG Isotype Control IgG1 (BD Biosciences), catalog number: 557273, subtype: 1)
Target substance 10: Mouse IgG antibody (mouse IgG Isotype Control IgG2a (BD Biosciences), catalog number: 553454, subtype: 2a)
Target substance 11: Mouse IgG antibody (mouse IgG Isotype Control IgG2b (manufactured by BD Biosciences), catalog number: 557351, subtype: 2b)
Target substance 12: Mouse IgG antibody (mouse IgG Isotype Control IgG3 (BD Biosciences), catalog number: 553486, subtype: 3)
（比較例１）
実施例２において、配列番号５及び６に示すＲＮＡの代わりに、配列番号１及び２の配列、並びに配列番号１〜４に示すＲＮＡのうち、シトシン残基及びウリジン残基をＤｕｒａｓｃｒｉｂｅ（登録商標） Ｔ７ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｋｉｔ（ＥＰＩＣＥＮＴＲＥ社製、カタログ番号：ＤＳ０１０９２５社製）を用いてフルオロ化した配列を用いた以外は、実施例２と同様に、結合の有無を観察した。その結果を表４に示す。なお、表４において、「１Ｆ」、「２Ｆ」、「３Ｆ」及び「４Ｆ」は、それぞれ、配列番号１〜４に示すＲＮＡを上記の通りフルオロ化したものを示す。
(Comparative Example 1)
In Example 2, in place of the RNAs shown in SEQ ID NOs: 5 and 6, the cytosine residues and uridine residues of the sequences shown in SEQ ID NOs: 1 and 2 and the RNAs shown in SEQ ID NOs: 1 to 4 were replaced with Durascribe (registered trademark). Existence of binding was observed in the same manner as in Example 2 except that a sequence fluorinated using T7 Transcription Kit (manufactured by EPICENTRE, catalog number: DS010925) was used. The results are shown in Table 4. In Table 4, “1F”, “2F”, “3F”, and “4F” indicate the RNAs shown in SEQ ID NOs: 1 to 4 that have been fluorinated as described above.
（実施例３）
比較例１において、配列番号１及び２並びにフルオロ化した各配列に代えて、配列番号１〜６に示すＲＮＡのうち、ウリジン残基のみをＤｕｒａｓｃｒｉｂｅ（登録商標） Ｔ７ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｋｉｔ（ＥＰＩＣＥＮＴＲＥ社製、カタログ番号：ＤＳ０１０９２５社製）を用いてフルオロ化した配列を用いた以外は、比較例１と同様に行い、結合の有無を観察した。その結果を表５に示す。なお、表５において、「１ＦＵ」、「２ＦＵ」、「３ＦＵ」、「４ＦＵ」、「５ＦＵ」及び「６ＦＵ」は、それぞれ、配列番号１〜６に示すＲＮＡを上記の通りフルオロ化したものを示す。
(Example 3)
In Comparative Example 1, in place of each of SEQ ID NOs: 1 and 2 and each of the fluorinated sequences, only the uridine residue in the RNAs shown in SEQ ID NOs: 1 to 6 was used as a Durascript (registered trademark) T7 Transfer Kit (manufactured by EPICENTRE, catalog No .: manufactured by DS010925 Co.) was used in the same manner as in Comparative Example 1 except that a sequence fluorinated was used, and the presence or absence of binding was observed. The results are shown in Table 5. In Table 5, “1FU”, “2FU”, “3FU”, “4FU”, “5FU”, and “6FU” are obtained by fluorinating the RNAs shown in SEQ ID NOs: 1 to 6 as described above. Show.
（比較例２）
実施例３において、フルオロ化した各配列に代えて、配列番号１〜６に示すＲＮＡのうち、シトシン残基のみをＤｕｒａｓｃｒｉｂｅ（登録商標） Ｔ７ Ｔｒａｎｓｃｒｉｐｔｉｏｎ Ｋｉｔ（ＥＰＩＣＥＮＴＲＥ社製、カタログ番号：ＤＳ０１０９２５社製）を用いてフルオロ化した配列を用いた以外は、実施例３と同様に行い、結合の有無を観察した。その結果を表６に示す。なお、表６において、「１ＦＣ」、「２ＦＣ」、「３ＦＣ」、「４ＦＣ」、「５ＦＣ」及び「６ＦＣ」は、それぞれ、配列番号１〜４に示すＲＮＡを上記の通りフルオロ化したものを示す。
(Comparative Example 2)
In Example 3, in place of each fluorinated sequence, only the cytosine residue in the RNAs shown in SEQ ID NOs: 1 to 6 was used for Duracribe (registered trademark) T7 Transcription Kit (manufactured by EPICENTRE, catalog number: DS010925) Except for using a sequence fluorinated using, the procedure was the same as in Example 3, and the presence or absence of binding was observed. The results are shown in Table 6. In Table 6, “1FC”, “2FC”, “3FC”, “4FC”, “5FC”, and “6FC” are those obtained by fluorinating the RNAs shown in SEQ ID NOS: 1-4 as described above. Show.
（製造例３）
製造例２において、配列番号１の代わりに、配列番号４を用いた以外は、製造例２と同様に、二次構造を予測した。その結果を図２に示す。
(Production Example 3)
In Production Example 2, a secondary structure was predicted in the same manner as in Production Example 2 except that SEQ ID NO: 4 was used instead of SEQ ID NO: 1. The result is shown in FIG.
このようにして得た配列番号４の二次構造の結果を参照すると、１６〜１９残基目及び４５〜４８残基目の塩基が４塩基対からなるステム長を有するステム領域を形成することが分かった。 Referring to the secondary structure result of SEQ ID NO: 4 obtained in this way, the bases at residues 16-19 and 45-48 form a stem region having a stem length of 4 base pairs. I understood.
製造例２において二次構造の予測を行った配列番号１とともに、本製造例３において二次構造の予測を行った配列番号４について、それぞれの二次構造に基づいて、種々のＲＮＡを合成した。 Various RNAs were synthesized based on the respective secondary structures of SEQ ID NO: 4 for which secondary structure was predicted in Production Example 2 and SEQ ID NO: 4 for which secondary structure was predicted in Production Example 3. .
まず、配列番号１の二次構造においてステム領域の末端側のいわゆるプライマー領域を欠失させ、グアノシン及びシチジンを用いて、ステム領域のステム長さを２つ延ばした配列番号８に示すＲＮＡを合成した。また、配列番号１の二次構造においてステム領域の末端側のいわゆるプライマー領域を欠失させ、ステム領域のステム長４の塩基対を、グアニン残基及びシトシン残基からなるステム長５のステム領域とした配列番号９、及びステム長６のステム領域とした配列番号１０を、それぞれ合成した。 First, in the secondary structure of SEQ ID NO: 1, a so-called primer region at the end of the stem region is deleted, and RNA shown in SEQ ID NO: 8 is synthesized using guanosine and cytidine and extending the stem length of the stem region by two. did. Further, in the secondary structure of SEQ ID NO: 1, a so-called primer region at the terminal end of the stem region is deleted, and a stem length 4 base pair of the stem region is replaced with a stem region of stem length 5 consisting of a guanine residue and a cytosine residue. SEQ ID NO: 9 and SEQ ID NO: 10 as a stem region having a stem length of 6 were synthesized.
一方、配列番号４の二次構造においてステム領域の末端側のいわゆるプライマー領域を欠失させ、このステム領域に代えて、グアノシン及びシチジンを用いて、ステム長さ４のステム領域を有する配列番号１１に示すＲＮＡを合成した。 On the other hand, in the secondary structure of SEQ ID NO: 4, a so-called primer region at the end of the stem region is deleted, and guanosine and cytidine are used in place of this stem region, and SEQ ID NO: 11 having a stem region of stem length 4 The RNA shown in Figure 1 was synthesized.
さらに、配列番号１の二次構造において、ステム領域の末端側のいわゆるプライマー領域を欠失させ、グアノシン及びシチジンを用いて、ステム領域のステム長さを２つ延ばしたステム長５の配列において、３’末端側のステム領域に隣接するシトシン残基を、グアニン残基に改変した、配列番号１２に示すＲＮＡを合成した。 Furthermore, in the secondary structure of SEQ ID NO: 1, in the sequence of stem length 5 in which a so-called primer region on the terminal side of the stem region is deleted, and the stem length of the stem region is extended by two using guanosine and cytidine, The RNA shown in SEQ ID NO: 12 was synthesized in which the cytosine residue adjacent to the 3 ′ terminal stem region was changed to a guanine residue.
またさらに、配列番号１の二次構造において、ステム領域の末端側のいわゆるプライマー領域を欠失させ、グアノシン及びシチジンを用いて、ステム領域のステム長さを２つ延ばしたステム長６の、配列番号１９に示すＲＮＡを合成した。また、配列番号４の二次構造において、ステム領域の末端側のいわゆるプライマー領域を欠失させ、グアノシン及びシチジンを用いて、ステム領域のステム長さを２つ延ばしたステム長６の、配列番号２０に示すＲＮＡを合成した。 Furthermore, in the secondary structure of SEQ ID NO: 1, a so-called primer region at the terminal end of the stem region is deleted, and a stem length 6 sequence is obtained by extending the stem length of the stem region by two using guanosine and cytidine RNA shown in No. 19 was synthesized. Further, in the secondary structure of SEQ ID NO: 4, a so-called primer region on the terminal side of the stem region is deleted, and the stem length 6 is obtained by extending the stem length of the stem region by 2 using guanosine and cytidine. RNA shown in 20 was synthesized.
（実施例４）
実施例２において、配列番号５〜７の代わりに、配列番号８〜１２及び１９〜２０を用いた以外は、実施例２と同様に行い、結合の有無を観察した。その結果を表７に示す。
Example 4
In Example 2, it carried out like Example 2 except having used sequence number 8-12 and 19-20 instead of sequence number 5-7, and the existence of binding was observed. The results are shown in Table 7.
（比較例３）
実施例４において、配列番号８〜１２及び１９〜２０の代わりに、配列番号１２において、５’末端側のステム領域に隣接するウリジン残基をグアニン残基に改変するとともに、３’末端側のステム領域に隣接するグアニン残基をアデニン残基に改変した配列番号１８の配列を用いた以外は、実施例４と同様に行い、結合の有無を観察した。その結果を表８に示す。
(Comparative Example 3)
In Example 4, instead of SEQ ID NOs: 8-12 and 19-20, in SEQ ID NO: 12, the uridine residue adjacent to the stem region on the 5 ′ end side is changed to a guanine residue, and at the 3 ′ end side Existence of binding was observed in the same manner as in Example 4 except that the sequence of SEQ ID NO: 18 in which the guanine residue adjacent to the stem region was changed to an adenine residue was used. The results are shown in Table 8.
（製造例４）
製造例１と同様に行い、配列番号２１〜２３を得た。
(Production Example 4)
It carried out like manufacture example 1 and obtained sequence number 21-23.
（実施例５）
実施例１において、配列番号１〜４の代わりに、配列番号２１〜２３を用い、且つ標的物質５〜８の代わりに、標的物質９を用いて行った以外は、実施例１と同様に行い、結合の有無を観察した。その結果を表９に示す。表９中、「＋」印は、結合の見られたものを、「△」印は、わずかに結合が見られたものを示す。
(Example 5)
In Example 1, it carried out similarly to Example 1 except having used sequence number 21-23 instead of sequence number 1-4, and using target substance 9 instead of target substance 5-8. The presence or absence of binding was observed. The results are shown in Table 9. In Table 9, a “+” mark indicates that a bond is observed, and a “Δ” mark indicates that a bond is slightly observed.
（製造例５）
安定化の目的のため、配列番号１１の塩基のうち、５’末端から２残基目のグアニン残基をシトシン残基に、３’末端から２残基目のシトシン残基をグアニン残基に、それぞれ改変した配列（以下、本製造例において、改変配列と称する。）を合成した。
(Production Example 5)
For the purpose of stabilization, among the bases of SEQ ID NO: 11, the second guanine residue from the 5 ′ end is the cytosine residue and the second cytosine residue from the 3 ′ end is the guanine residue. Each of the modified sequences (hereinafter referred to as a modified sequence in this production example) was synthesized.
得た改変配列の５’末端を、定法に従いビオチン化して、配列番号２４に示すＲＮＡを得た。 The 5 ′ end of the obtained modified sequence was biotinylated according to a conventional method to obtain RNA shown in SEQ ID NO: 24.
また、改変配列の５’末端側にデオキシアデニンを５残基付加した配列を合成し、その５’末端を、上記と同様にビオチン化して、配列番号２５に示すＲＮＡを得た。 Further, a sequence in which 5 residues of deoxyadenine were added to the 5 ′ end side of the modified sequence was synthesized, and the 5 ′ end was biotinylated in the same manner as described above to obtain RNA shown in SEQ ID NO: 25.
また、改変配列の３’末端側にデオキシアデニンを１０残基付加した配列を合成し、その３’末端を、上記と同様にビオチン化して、配列番号２６に示すＲＮＡを得た。 Further, a sequence in which 10 residues of deoxyadenine was added to the 3 ′ end side of the modified sequence was synthesized, and the 3 ′ end was biotinylated in the same manner as described above to obtain RNA shown in SEQ ID NO: 26.
また、改変配列の５’末端側に、炭素数１８からなる炭素鎖（商品名; SPACER18, GLEN RESERCH社，品番10-1918-90）を３つ付加した配列を合成し、その５’末端を、上記と同様にビオチン化して、配列番号２７に示すＲＮＡを得た。 In addition, a sequence in which three carbon chains having 18 carbon atoms (trade name: SPACER18, GLEN RESERCH, product number 10-1918-90) are added to the 5 ′ end side of the modified sequence, and the 5 ′ end is synthesized. In the same manner as above, biotinylation was performed to obtain RNA shown in SEQ ID NO: 27.
さらに、改変配列の３’末端を、定法に従いビオチン化して、配列番号２８に示すＲＮＡを得た。 Furthermore, the 3 ′ end of the modified sequence was biotinylated according to a conventional method to obtain RNA shown in SEQ ID NO: 28.
また、改変配列の３’末端側にデオキシアデニンを５残基付加した配列を合成し、その３’末端を、上記と同様にビオチン化して、配列番号２９に示すＲＮＡを得た。 Further, a sequence in which 5 residues of deoxyadenine were added to the 3 ′ end side of the modified sequence was synthesized, and the 3 ′ end was biotinylated in the same manner as described above to obtain RNA shown in SEQ ID NO: 29.
また、改変配列の５’末端側にデオキシアデニンを１０残基付加した配列を合成し、その５’末端を、上記と同様にビオチン化して、配列番号３０に示すＲＮＡを得た。 Further, a sequence in which 10 residues of deoxyadenine was added to the 5 ′ end side of the modified sequence was synthesized, and the 5 ′ end was biotinylated in the same manner as described above to obtain RNA shown in SEQ ID NO: 30.
また、改変配列の３’末端側に、上記と同様に炭素数１８からなる炭素鎖を３つ付加した配列を合成し、その５’末端を、上記と同様にビオチン化して、配列番号３１に示すＲＮＡを得た。 Further, a sequence in which three carbon chains having 18 carbon atoms were added to the 3 ′ end side of the modified sequence was synthesized in the same manner as described above, and the 5 ′ end was biotinylated in the same manner as described above to obtain SEQ ID NO: 31. The indicated RNA was obtained.
（実施例６）
実施例１において、配列番号１〜４に代えて、配列番号２４〜２７を用いた以外は、実施例１と同様に行い、各配列の結合定数（Ｋｄ値）を算出した。その結果を表１０に示す。
(Example 6)
In Example 1, except that SEQ ID NOS: 24-27 were used instead of SEQ ID NOS: 1-4, the procedure was performed in the same manner as in Example 1, and the binding constant (Kd value) of each sequence was calculated. The results are shown in Table 10.
（実施例７）
実施例６において、配列番号２４〜２７のうち、配列番号２５を用い、２００ｎＭ、１００ｎＭ、５０ｎＭ、２５ｎＭ、１２．５ｎＭ及び６．２５ｎＭのマウスＩｇＧ抗体の代わりに、２００ｎＭ、１００ｎＭ、５０ｎＭ、２５ｎＭ、１２．５ｎＭ及び６．２５ｎＭの標的物質９、１０及び１２を用いた以外は、実施例６と同様に行い、この配列の各標的物質に対する結合定数（Ｋｄ）を算出した。その結果を表１１に示す。
(Example 7)
In Example 6, using SEQ ID NO: 25 out of SEQ ID NO: 24-27, instead of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM mouse IgG antibodies, 200 nM, 100 nM, 50 nM, 25 nM, Except that 12.5 nM and 6.25 nM of target substances 9, 10 and 12 were used, the procedure was carried out in the same manner as in Example 6, and the binding constant (Kd) for each target substance of this sequence was calculated. The results are shown in Table 11.
（実施例８）
９６ウェルのＥＬＩＳＡプレート（商品名：Ｉｍｍｕｎｏ ９６ Ｍｉｃｒｏｗｅｌｌ Ｐｌａｔｅｓ ＭａｘｉＳｏｒｐ（Ｎｕｎｃ社製）、カタログ番号：４３０３４１）に、４００ｎｇの抗ＦＬＡＧマウスＩｇＧ抗体（標的物質２）を有する５０μＬのＰＢＳ溶液（ＰＢＳ：８．１ｍＭ Ｎａ２ＨＰＯ４、１．４７ｍＭ ＫＨ２ＰＯ４、１３７ｍＭ ＮａＣｌ、２．６８ｍＭ ＫＣｌ（Ｎｉｐｐｏｎ Ｇｅｎｅ社製、カタログ番号：３１４−９０８１５））を各ウェルに分注し、４℃で一昼夜静置した。その後、ブロッキング溶液（ＰＢＳ、０．０５％ Ｔｗｅｅｎ２０、５ｍＭ ＭｇＣｌ２、１×デンハルト溶液及び０．１％ アセチル化ＢＳＡ）１００μＬを各ウェルに分注し、１時間、室温で静置し、その後、ウェルの液を取り除いた。
(Example 8)
A 96-well ELISA plate (trade name: Immuno 96 Microwell Plates MaxiSorp (manufactured by Nunc), catalog number: 430341) with 400 ng of an anti-FLAG mouse IgG antibody (target substance 2) (PBS: 8. 1 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 , 137 mM NaCl, 2.68 mM KCl (manufactured by Nippon Gene, catalog number: 314-90815)) were dispensed into each well and left at 4 ° C. overnight. . Thereafter, 100 μL of blocking solution (PBS, 0.05% Tween 20, 5 mM MgCl 2 , 1 × Denhardt's solution and 0.1% acetylated BSA) was dispensed into each well, and allowed to stand at room temperature for 1 hour. Well fluid was removed.
その後、配列番号２４〜２７及び配列番号２８〜３１の配列からなるＲＮＡであって、９５度３分間の加熱後室温にて除冷しリフォールディングさせたもの（３００ｎＭ）、及びストレプトアビジン−ＨＲＰコンジュゲート（商品名ＥＣＬ Ｓｔｒｅｐｔａｖｉｄｉｎ−ＨＲＰ Ｃｏｎｊｕｇａｔｅ、ＧＥヘルスケアバイオサイエンス社製、カタログ番号：ＲＰＮ２１９５）（１／５００）を有するＰＢＳ−Ｔ＋Ｍｇ溶液（ＰＢＳ、０．０５％ Ｔｗｅｅｎ２０及び５ｍＭ ＭｇＣｌ２）であるＲＮＡ溶液５０μＬを各ウェルに分注し、３０分間、室温で静置した。 Thereafter, RNA comprising the sequences of SEQ ID NO: 24-27 and SEQ ID NO: 28-31, which was heated at 95 ° C. for 3 minutes, cooled at room temperature and refolded (300 nM), and streptavidin-HRP conjugate RNA that is a PBS-T + Mg solution (PBS, 0.05% Tween 20 and 5 mM MgCl 2 ) having a gate (trade name ECL Streptavidin-HRP Conjugate, manufactured by GE Healthcare Biosciences, catalog number: RPN2195) (1/500) 50 μL of the solution was dispensed into each well and allowed to stand at room temperature for 30 minutes.
その後、１００μＬのＰＢＳ−Ｔ＋Ｍｇ溶液を用いて３回各ウェルを洗浄し、各ウェルに１００μＬのＨＲＰ基質（商品名：１−Ｓｔｅｐ Ｔｕｒｂｏ ＴＭＢ−ＥＬＩＳＡ （ＰＩＥＲＣＥ社製）、カタログ番号：３４０２２））を加え、３０分間、室温で反応させ、その後、１ＭのＨ２ＳＯ４で反応を停止した。各ウェルの４５０ｎｍにおける吸光度（参照波長は６２０ｎｍ）を測定した。その結果を図３に示す。 Thereafter, each well was washed three times with 100 μL of PBS-T + Mg solution, and 100 μL of HRP substrate (trade name: 1-Step Turbo TMB-ELISA (manufactured by PIERCE), catalog number: 34002)) was added to each well. In addition, the reaction was allowed to proceed for 30 minutes at room temperature, after which the reaction was quenched with 1M H 2 SO 4 . Absorbance of each well at 450 nm was measured (reference wavelength was 620 nm). The result is shown in FIG.
図３において、縦軸は、４５０ｎｍの測定波長、６２０ｎｍの参照波長を用いて得た吸光度を示し、横軸は、ＲＮＡ溶液に含まれるＲＮＡに対応する配列番号の番号を示す。また、「ＮＣ」は、ＲＮＡ溶液において、ＲＮＡを有さないものを用いて得た結果を示し、「ＰＣ」は、標的物質２を有するＰＢＳ溶液を用いて一昼夜行う反応を行わずに、且つＨＲＰ基質を用いた反応を行う際に、ストレプトアビジン−ＨＲＰコンジュゲート（商品名ＥＣＬ Ｓｔｒｅｐｔａｖｉｄｉｎ−ＨＲＰ Ｃｏｎｊｕｇａｔｅ、ＧＥヘルスケアバイオサイエンス社製、カタログ番号：ＲＰＮ２１９５）（１／５００）を有するＰＢＳ−Ｔ＋Ｍｇ溶液を添加した得た結果を示す。 In FIG. 3, the vertical axis indicates the absorbance obtained using a measurement wavelength of 450 nm and a reference wavelength of 620 nm, and the horizontal axis indicates the number of the SEQ ID NO corresponding to the RNA contained in the RNA solution. In addition, “NC” indicates a result obtained using an RNA solution that does not have RNA, and “PC” indicates that a reaction that is performed day and night using a PBS solution containing the target substance 2 is not performed, and PBS-T + Mg solution having streptavidin-HRP conjugate (trade name ECL Streptavidin-HRP Conjugate, manufactured by GE Healthcare Biosciences, catalog number: RPN2195) (1/500) when performing a reaction using an HRP substrate The result obtained by adding is shown.
（実施例９）
実施例８において、下記（Ｉ）〜（Ｖ）の通りとした以外は、実施例８と同様に行い、４５０ｎｍの測定波長、６２０ｎｍの参照波長を用いた吸光度を得た。
Example 9
Example 8 was carried out in the same manner as Example 8 except that the following (I) to (V) were used, and absorbance was obtained using a measurement wavelength of 450 nm and a reference wavelength of 620 nm.
（Ｉ）抗ＦＬＡＧマウスＩｇＧ抗体（標的物質２）の代わりに、下記（１）〜（６）の各ＩｇＧを用いた
（ＩＩ）配列番号２４〜２７及び配列番号２８〜３１の配列からなるＲＮＡの代わりに、配列番号２８に示す配列からなるＲＮＡを用いた
（ＩＩＩ）リフォールディングさせたＲＮＡの濃度を５０ｎＭとした
（ＩＶ）ストレプトアビジン−ＨＲＰコンジュゲート（１／５００）に代えて、ストレプトアビジン−ＨＲＰコンジュゲート（１／１０００）を用いた
（Ｖ）ＲＮＡ溶液を各ウェルに分注して室温で静置する時間を２０分間とした
(I) Instead of the anti-FLAG mouse IgG antibody (target substance 2), each IgG of the following (1) to (6) was used. (II) RNA comprising the sequences of SEQ ID NOs: 24-27 and 28-31 (III) The concentration of refolded RNA was set to 50 nM instead of (IV) Streptavidin-HRP conjugate (1/500) instead of streptavidin -HRP conjugate (1/1000) was used. (V) RNA solution was dispensed into each well and allowed to stand at room temperature for 20 minutes.
（１）マウス抗ＦＬＡＧ Ｍ２抗体（標的物質２）（図４中、マウスと表記）
（２）ウサギ抗ＧＳＴポリクローナル抗体（ケミコン社製、カタログ番号：ＡＢ３２８２）（図４中、ウサギと表記）
（３）ラットＩｇＧ（Ｓａｎｔａ Ｃｒｕｚ社製、カタログ番号：ｓｃ−２０２６）（図４中、ラットと表記）
（４）ヤギ抗ウサギＩｇＧ（ＣＨＥＭＩＣＯＮ社製、カタログ番号：ＡＰ−１３２）（図４中、ヤギと表記）
（５）ヒト抗ＩｇＧ（Ｂｅｔｈｙｌ社製、カタログ番号：Ｐ８０−１０４）（図４中、ヒトと表記）
（６）モルモットＩｇＧ（Ｊａｃｋｓｏｎ Ｉｍｍｕｎｏ Ｒｅｓｅａｒｃｈ社製、カタログ番号：００６−０００−００２）（図４中、モルモットと表記）
(1) Mouse anti-FLAG M2 antibody (target substance 2) (indicated as mouse in FIG. 4)
(2) Rabbit anti-GST polyclonal antibody (Chemicon, catalog number: AB3282) (indicated as rabbit in FIG. 4)
(3) Rat IgG (manufactured by Santa Cruz, catalog number: sc-2026) (indicated as rat in FIG. 4)
(4) Goat anti-rabbit IgG (manufactured by Chemicon, catalog number: AP-132) (shown as goat in FIG. 4)
(5) Human anti-IgG (manufactured by Bethyl, catalog number: P80-104) (indicated as human in FIG. 4)
(6) Guinea pig IgG (Jackson Immuno Research, catalog number: 006-000-002) (denoted as guinea pig in FIG. 4)
また、配列番号２８に示す配列からなるＲＮＡに代えて、ヒツジ由来の抗マウスＩｇＧ−ＨＲＰ結合完全抗体（ＧＥ社製、カタログ番号：Ｎ９３１）をＰＢＳ−Ｔ＋Ｍｇで２０００倍に希釈したものを用いて、上記と同様に吸光度を得た。その結果を図５に示す。なお、図４及び５において、「ＮＣ」及び「ＰＣ」は、図３と同様である。 Further, instead of RNA having the sequence shown in SEQ ID NO: 28, a sheep-derived anti-mouse IgG-HRP-conjugated complete antibody (GE, catalog number: N931) diluted 2000 times with PBS-T + Mg was used. Absorbance was obtained in the same manner as above. The result is shown in FIG. 4 and 5, “NC” and “PC” are the same as those in FIG.
（実施例１０）
実施例９において、下記（Ｉ）〜（ＩＩＩ）の通りに変更した以外は、実施例９と同様に行い、４５０ｎｍの測定波長、６２０ｎｍの参照波長を用いた吸光度を得た。
(Example 10)
In Example 9, except having changed as follows (I)-(III), it carried out similarly to Example 9 and obtained the light absorbency using the measurement wavelength of 450 nm, and the reference wavelength of 620 nm.
（Ｉ）上記（１）〜（６）の各ＩｇＧに代えて、下記表１２に記載の（１）〜（１４）の各ＩｇＧを用いた
（ＩＩ）ＲＮＡ溶液中のＲＮＡの濃度を２００ｎＭとした
（ＩＩＩ）各ウェルにＨＲＰ基質を添加して室温で反応させる時間を５０分間とした
(I) Instead of each IgG of (1) to (6) above, each IgG of (1) to (14) described in Table 12 below was used. (II) The RNA concentration in the RNA solution was 200 nM. (III) The time for adding HRP substrate to each well and reacting at room temperature was 50 minutes.
その結果を図６に示す。なお、図６において、「ＮＣ」及び「ＰＣ」は、図３と同様である。 The result is shown in FIG. In FIG. 6, “NC” and “PC” are the same as those in FIG.
（実施例１１）
実施例９において、下記（Ｉ）〜（ＩＶ）の通りとした以外は、実施例９と同様に行い、４５０ｎｍの測定波長、６２０ｎｍの参照波長を用いた吸光度を得た。
(Example 11)
Example 9 was carried out in the same manner as Example 9 except that the following (I) to (IV) were used, and absorbance was obtained using a measurement wavelength of 450 nm and a reference wavelength of 620 nm.
（Ｉ）４００ｎｇの標的物質２に代えて、１０００ｎｇ、１００ｎｇ、１０ｎｇ、１ｎｇ、０．１ｎｇ、０．００１ｎｇ及び０．０００１ｎｇの標的物質２を用いた
（ＩＩ）リフォールディングさせたＲＮＡの濃度を５０ｎＭとした
（ＩＩＩ）ストレプトアビジン−ＨＲＰコンジュゲート（１／５００）に代えて、ストレプトアビジン−ＨＲＰコンジュゲート（１／１０００）を用いた
（ＩＶ）ＲＮＡ溶液を各ウェルに分注して室温で静置する時間を２０分間とした
(I) In place of 400 ng of target substance 2, 1000 ng, 100 ng, 10 ng, 1 ng, 0.1 ng, 0.001 ng and 0.0001 ng of target substance 2 were used. (II) The concentration of refolded RNA was 50 nM. (III) Instead of streptavidin-HRP conjugate (1/500), streptavidin-HRP conjugate (1/1000) was used. (IV) RNA solution was dispensed into each well and allowed to stand at room temperature. The setting time was 20 minutes
その結果を図７に示す。なお、図７において、横軸は、標的物質２の量を示し、縦軸は、４５０ｎｍの測定波長、６２０ｎｍの参照波長を用いて得た吸光度を示す。 The result is shown in FIG. In FIG. 7, the horizontal axis indicates the amount of the target substance 2, and the vertical axis indicates the absorbance obtained using a measurement wavelength of 450 nm and a reference wavelength of 620 nm.
また、標的物質２８に示す配列からなるＲＮＡに代えて、ヒツジ由来の抗マウスＩｇＧ−ＨＲＰ結合完全抗体（ＧＥ社製、カタログ番号：Ｎ９３１）をＰＢＳ−Ｔ＋Ｍｇで２０００倍に希釈したものを用いて、上記と同様に吸光度を得た。その結果を図８に示す。図８において、横軸は、ＩｇＧ抗体の量を示し、縦軸は、図７と同様である。 Further, instead of RNA consisting of the sequence shown in the target substance 28, a sheep-derived anti-mouse IgG-HRP-conjugated complete antibody (manufactured by GE, catalog number: N931) diluted 2000 times with PBS-T + Mg was used. Absorbance was obtained in the same manner as above. The result is shown in FIG. In FIG. 8, the horizontal axis indicates the amount of IgG antibody, and the vertical axis is the same as in FIG.
（実施例１２）
実施例８において、下記（Ｉ）〜（ＩＩＩ）の通りとした以外は、実施例８と同様に行い、４５０ｎｍの測定波長、６２０ｎｍの参照波長を用いて得た吸光度を得た。
Example 12
Example 8 was carried out in the same manner as in Example 8 except that the following (I) to (III) were used, and the absorbance obtained using a measurement wavelength of 450 nm and a reference wavelength of 620 nm was obtained.
（Ｉ）配列番号２４〜２７及び配列番号２８〜３１の配列からなるＲＮＡであって、９５度３分間の加熱後室温にて除冷しリフォールディングさせたもの（３００ｎＭ）に代えて、８００ｎＭ、４００ｎＭ、２００ｎＭ、１００ｎＭ、５０ｎＭ、２５ｎＭ、１２．５ｎＭ及び６．２５ｎＭの配列番号２５の配列からなるＲＮＡを用いた
（ＩＩ）ストレプトアビジン−ＨＲＰコンジュゲート（１／５００）に代えて、ストレプトアビジン−ＨＲＰコンジュゲート（１／１０００）を用いた
（ＩＩＩ）ＲＮＡ溶液を各ウェルに分注して室温で静置する時間を２０分間とした
(I) RNA consisting of the sequences of SEQ ID NO: 24-27 and SEQ ID NO: 28-31, which was heated at 95 ° C. for 3 minutes, then cooled at room temperature and refolded (300 nM), instead of 800 nM, (II) Streptavidin-HRP conjugate (1/500) was used instead of streptavidin-HRP conjugate (1/500) using 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM RNA consisting of the sequence number 25 Using HRP conjugate (1/1000) (III) RNA solution was dispensed into each well and allowed to stand at room temperature for 20 minutes
その結果を図９に示す。図９において、横軸は、用いたＲＮＡの濃度を示し、縦軸は、図３と同様である。 The result is shown in FIG. In FIG. 9, the horizontal axis indicates the concentration of RNA used, and the vertical axis is the same as in FIG.
（実施例１３）
ＳＡ−Ａｇａｒｏｓｅ（商品名：ＵｌｔｒａＬｉｎｋ（登録商標）Ｉｍｍｏｂｉｌｉｚｅｄ Ｓｔｒｅｐｔａｖｉｄｉｎ Ｐｌｕｓ（ＰＩＥＲＣＥ社製）、カタログ番号：５３１１７）をカラム（Ｍｉｃｒｏ Ｂｉｏ−ｓｐｉｎ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ ｃｏｌｕｍｎｓ （Ｂｉｏ−ｒａｄ社製、カタログ番号：７３２−６２０４））に充填し、ＰＢＳ溶液で２回洗浄した。
(Example 13)
SA-Agarose (trade name: UltraLink (registered trademark) Immobilized Streptavidin Plus (manufactured by PIERCE), catalog number: 53117) and column (Micro Bio-spin Chromatography columns (manufactured by Bio-rad 2-), catalog number: 73) And washed twice with PBS solution.
一方、配列番号２５に示すＲＮＡとＰＢＳ−Ｍｇ溶液とからなるＲＮＡ溶液（ＲＮＡ濃度１０μＭ）を調製した。 On the other hand, an RNA solution (RNA concentration 10 μM) composed of the RNA shown in SEQ ID NO: 25 and a PBS-Mg solution was prepared.
上記のＳＡ−Ａｇａｒｏｓｅを有するカラムに、このＲＮＡ溶液を添加し、４℃で１時間、インキュベーションした後、ＰＢＳ溶液２５０μＬを用いて、３回洗浄した。 This RNA solution was added to the above-mentioned column having SA-Agarose, incubated at 4 ° C. for 1 hour, and then washed 3 times with 250 μL of PBS solution.
このようにして調製したカラムに、マウス腹水（Ｃｏｎｔｒｏｌ ｍｏｕｓｅ ａｓｃｉｔｅｓ ｆｌｕｉｏｄ Ｃｌｏｎｅ ＮＳ−１（ＳＩＧＭＡ社製）、カタログ番号：Ｍ８２７３、２３ｍｇ／ｍｌタンパク）と標的物質２に示すマウス抗ＦＬＡＧ抗体とを有するＰＢＳ−Ｍｇ溶液（抗体濃度１ｍｇ／ｍＬ）（以下、腹水−抗ＦＬＡＧ抗体混合液と称する。）を添加し、室温で３０分間、インキュベートした。 PBS having mouse ascites (Control mouse assays fluid Clone NS-1 (manufactured by SIGMA), catalog number: M8273, 23 mg / ml protein) and mouse anti-FLAG antibody shown in target substance 2 on the column thus prepared -Mg solution (antibody concentration 1 mg / mL) (hereinafter referred to as an ascites-anti-FLAG antibody mixture) was added and incubated at room temperature for 30 minutes.
このようにして得たカラムを、遠心分離し、上清を回収し、素通り画分とした。さらにこのカラムに、ＰＢＳ溶液２５０μＬで３回洗浄し、この洗浄液を全て回収した。なお、これらの洗浄液をそれぞれ、洗浄液１、２及び３と称する。 The column thus obtained was centrifuged, and the supernatant was collected and used as a flow-through fraction. Further, this column was washed with 250 μL of PBS solution three times, and all of this washing solution was recovered. These cleaning liquids are referred to as cleaning liquids 1, 2, and 3, respectively.
次に、このカラムに、５０μＬのＰＢＳ溶液を添加して、室温で１０分間インキュベートし、カラムを遠心分離して、溶出液１を得た。また、このカラムに、上記と同様にＰＢＳ溶液を用いて、インキュベーション及び遠心分離を行い、溶出液２を得た。 Next, 50 μL of PBS solution was added to this column, incubated at room temperature for 10 minutes, and the column was centrifuged to obtain eluate 1. Further, this column was incubated and centrifuged using a PBS solution in the same manner as described above to obtain an eluate 2.
その後、このカラムに、５０μＬのＰＢＳ−ＥＤＴＡ溶液（ＰＢＳ及び１０ｍＭ ＥＤＴＡ）を添加して、室温で１０分間インキュベートし、カラムを遠心分離して、溶出液３を得た。また、このカラムに上記と同様にＰＢＳ−ＥＤＴＡ溶液を用いて、インキュベーション及び遠心分離を行い、溶出液４を得た。 Thereafter, 50 μL of PBS-EDTA solution (PBS and 10 mM EDTA) was added to this column, incubated at room temperature for 10 minutes, and the column was centrifuged to obtain eluate 3. Further, the column was incubated and centrifuged using a PBS-EDTA solution in the same manner as described above to obtain an eluate 4.
その後、このカラムに、５０μＬのＰＢＳ−ＲＮａｓｅＡ（ＰＢＳ＋０．０１ｍｇ／ｍＬ ＲＮａｓｅＡ）を添加して、室温で１０分間インキュベートし、カラムを遠心分離して、溶出液５を得た。 Thereafter, 50 μL of PBS-RNase A (PBS + 0.01 mg / mL RNase A) was added to the column, incubated at room temperature for 10 minutes, and the column was centrifuged to obtain eluate 5.
このようにして得た各サンプルを、ＳＤＳサンプルバッファーに加え、９５℃で５分間加熱し、ＳＤＳ−ＰＡＧＥ用サンプルとした。これを、５〜２０％のＳＤＳ−ＰＡＧＥ用ゲルにアプライし、３０分間泳動し、染色液（ＧｅｌＣｏｄｅ Ｂｌｕｅ Ｓｔａｉｎ Ｒｅａｇｅｎｔ（ＰＩＥＲＣＥ社製、カタログ番号：２４５９０））で染色し、ＳＤＳ−ＰＡＧＥ像を得た。その結果を図１０に示す。なお、図１０において、各レーンは、下記に示す通りである。 Each sample thus obtained was added to the SDS sample buffer and heated at 95 ° C. for 5 minutes to obtain a sample for SDS-PAGE. This was applied to a 5-20% SDS-PAGE gel, run for 30 minutes, and stained with a staining solution (GelCode Blue Stain Reagent (manufactured by PIERCE, catalog number: 24590)) to obtain an SDS-PAGE image. It was. The result is shown in FIG. In FIG. 10, each lane is as shown below.
Ｍ：Ｍａｒｋｅｒ（Ｐｒｅｃｉｓｉｏｎ Ｐｌｕｓ Ｐｒｏｔｅｉｎ Ｋａｌｅｉｄｏｓｃｏｐｅ Ｓｔａｎｄａｒｄｓ（Ｂｉｏ−ｒａｄ社製）、カタログ番号：１６１−０３７５）
１：腹水−抗ＦＬＡＧ抗体混合液 １μＬ（１１．５μｇｌ ｐｒｏｔｅｉｎ ＋ １μｇ ａｎｔｉ−ＦＬＡＧ ＭＡｂ相当）
２：素通り画分１μＬ （腹水−抗ＦＬＡＧ抗体混合液のカラム素通り画分ｔｏｔａｌ ５０μＬ）
３：洗浄液３ ５μＬ（カラムｗａｓｈ ｔｏｔａｌ ２５０μＬ）
４：溶出液１ ５μＬ（ＰＢＳ；Ｍｇ（−）での溶出画分 ｔｏｔａｌ ５０μＬ）
４：溶出液２ ５μＬ（ＰＢＳ；Ｍｇ（−）での溶出画分 ｔｏｔａｌ ５０μＬ）
６：溶出液３ ５μＬ（ＰＢＳ；ＥＤＴＡ（＋）での溶出画分 ｔｏｔａｌ ５０μＬ）
７：溶出液４ ５μＬ（ＰＢＳ；ＥＤＴＡ（＋）での溶出画分 ｔｏｔａｌ ５０μＬ）
８：溶出液５ ５μＬ（ＰＢＳ；ＲＮＡ（＋）での溶出画分 ｔｏｔａｌ ５０μＬ）
９：溶出終了後のＳＡ−Ａｇａｒｏｓｅ ５μＬ（溶出後のＳＡ−ａｇａｒｏｓｅ−ｍｏｕｓｅ ＩｇＧアプタマー複合体 ｔｏｔａｌ ５０μＬ）
１０：抗ＦＬＡＧ抗体 １μＬ（１ｍｇ／ｍｌ １μｇ相当）
M: Marker (Precision Plus Protein Kaleidoscope Standards (manufactured by Bio-rad), catalog number: 161-0375)
1: Ascites-anti-FLAG antibody mixed solution 1 μL (11.5 μg protein + 1 μg anti-FLAG MAb equivalent)
2: Flow-through fraction 1 μL (ascites-anti-FLAG antibody mixed solution column flow-through total 50 μL)
3: Washing liquid 3 5 μL (column wash total 250 μL)
4: 5 μL of eluate 1 (PBS; fraction eluted with Mg (−) total 50 μL)
4: 5 μL of eluate 2 (PBS; elution fraction with Mg (−) total 50 μL)
6: 5 μL of eluate 3 (PBS; fraction eluted with EDTA (+) total 50 μL)
7: Eluent 4 5 μL (PBS; fraction eluted with EDTA (+) total 50 μL)
8: 5 μL of eluate 5 (PBS; fraction eluted with RNA (+) total 50 μL)
9: SA-Agarose 5 μL after elution (SA-agarose-mouse IgG aptamer complex 50 μL after elution)
10: 1 μL of anti-FLAG antibody (equivalent to 1 μg of 1 mg / ml)
（実施例１４）
１０ｍｇ／ｍＬのＤｙｎａｂｅａｄｓ Ｍ−２８０ ｓｔｒｅｐｔａｖｉｄｉｎ（Ｉｎｖｉｔｒｏｇｅｎ社製、カタログ番号：１１２．０５Ｄ）２５０μＬを１．５ｍＬチューブに移し、ＭａｇｎａＲａｃｋ（Ｉｎｖｉｔｒｏｇｅｎ社製、カタログ番号：ＣＳ１５０００）に立て、上清を回収した後、ＰＢＳ−Ｍｇ溶液（ＰＢＳ及び５ｍＭ ＭｇＣｌ２）で２回洗浄した。
(Example 14)
After transferring 250 μL of 10 mg / mL Dynabeads M-280 streptavidin (manufactured by Invitrogen, catalog number: 112.05D) to a 1.5 mL tube, standing on MagnaRack (manufactured by Invitrogen, catalog number: CS15000), and collecting the supernatant And washed twice with PBS-Mg solution (PBS and 5 mM MgCl 2 ).
一方、配列番号２５及び２６に示すＲＮＡを有するＰＢＳ−Ｍｇ溶液（ＲＮＡ濃度１０μＭ）を調製した。 On the other hand, a PBS-Mg solution (RNA concentration 10 μM) having RNAs shown in SEQ ID NOs: 25 and 26 was prepared.
上記のＤｙｎａｂｅａｄｓを有する液に、このＲＮＡ溶液を添加し、４℃で１時間、インキュベーションした後、ＰＢＳ−Ｍｇ５００μＬを用いて、３回洗浄した。 This RNA solution was added to the solution having the above Dynabeads, incubated at 4 ° C. for 1 hour, and then washed 3 times with 500 μL of PBS-Mg.
このようにして調製したＤｙｎａｂｅａｄｓに、マウス腹水（Ｃｏｎｔｒｏｌ ｍｏｕｓｅ ａｓｃｉｔｅｓ ｆｌｕｉｏｄ Ｃｌｏｎｅ ＮＳ−１（ＳＩＧＭＡ社製）、カタログ番号：Ｍ８２７３、２３ｍｇ／ｍｌタンパク）と標的物質２に示すマウス抗ＦＬＡＧ抗体を有するＰＢＳ−Ｍｇ−Ｉ溶液（抗体濃度１ｍｇ／ｍＬ）とを有する腹水−抗ＦＬＡＧ抗体混合液を添加し、４℃で１５分間、インキュベートした。 PBS- having mouse anti-FLAG antibody shown in Target substance 2 and Dynabeads prepared in this manner were added to mouse ascites (Control mouse assays fluone Clone NS-1 (manufactured by SIGMA), catalog number: M8273, 23 mg / ml protein). Ascites-anti-FLAG antibody mixture with Mg-I solution (antibody concentration 1 mg / mL) was added and incubated at 4 ° C. for 15 minutes.
このようにして得たＤｙｎａｂｅａｄｓを、遠心分離し、上清を回収し、素通り画分とした。さらにこのＤｙｎａｂｅａｄｓに、ＰＢＳ−Ｍｇ溶液で３回洗浄し、この洗浄液を全て回収した。なお、これらの洗浄液をそれぞれ、洗浄液１、２及び３と称する。 The Dynabeads thus obtained were centrifuged and the supernatant was collected and used as a flow-through fraction. Further, this Dynabeads was washed three times with a PBS-Mg solution, and all the washings were collected. These cleaning liquids are referred to as cleaning liquids 1, 2, and 3, respectively.
次に、このＤｙｎａｂｅａｄｓに、５０μＬのＰＢＳ溶液を添加して、４℃で１０分間インキュベートし、カラムを遠心分離して、溶出液１を得た。さらに、このＤｙｎａｂｅａｄｓに、５０μＬのＰＢＳ−ＥＤＴＡ溶液（ＰＢＳ、１０ｍＭ ＥＤＴＡ及び１×ＰｒｏｔｅｃｔＲＮＡ（登録商標）ＲＮａｓｅ Ｉｎｈｉｂｉｔｏｒ（同前））を添加して、４℃で１０分間インキュベートし、カラムを遠心分離して、溶出液２を得た。 Next, 50 μL of PBS solution was added to this Dynabeads, incubated at 4 ° C. for 10 minutes, and the column was centrifuged to obtain eluate 1. Furthermore, 50 μL of PBS-EDTA solution (PBS, 10 mM EDTA and 1 × ProtectRNA (registered trademark) RNase Inhibitor (same as above)) was added to the Dynabeads, incubated at 4 ° C. for 10 minutes, and the column was centrifuged. Thus, an eluate 2 was obtained.
このようにして得た各サンプルを、ＳＤＳサンプルバッファーに加え、９５℃で５分間加熱し、ＳＤＳ−ＰＡＧＥ用サンプルとした。これを、５〜２０％のＳＤＳ−ＰＡＧＥ用ゲルにアプライし、３０分間泳動し、染色液（ＧｅｌＣｏｄｅ Ｂｌｕｅ Ｓｔａｉｎ Ｒｅａｇｅｎｔ（ＰＩＥＲＣＥ社製、カタログ番号：２４５９０））で染色し、ＳＤＳ−ＰＡＧＥ像を得た。その結果を図１１に示す。なお、図１１において、各レーンは、下記に示す通りである。 Each sample thus obtained was added to the SDS sample buffer and heated at 95 ° C. for 5 minutes to obtain a sample for SDS-PAGE. This was applied to a 5-20% SDS-PAGE gel, run for 30 minutes, and stained with a staining solution (GelCode Blue Stain Reagent (manufactured by PIERCE, catalog number: 24590)) to obtain an SDS-PAGE image. It was. The result is shown in FIG. In FIG. 11, each lane is as shown below.
Ｍ：Ｍａｒｋｅｒ（Ｐｒｅｃｉｓｉｏｎ Ｐｌｕｓ Ｐｒｏｔｅｉｎ Ｋａｌｅｉｄｏｓｃｏｐｅ Ｓｔａｎｄａｒｄｓ（Ｂｉｏ−ｒａｄ社製）、カタログ番号：１６１−０３７５）
１：腹水−抗ＦＬＡＧ抗体混合液 １μＬ（１１．５μｇｌ ｐｒｏｔｅｉｎ ＋ １μｇ ａｎｔｉ−ＦＬＡＧ ＭＡｂ相当）
２：溶出液１ ４０μＬ相当
（ＰＢＳのみでの溶出画分 ｔｏｔａｌ ５０μＬのうち４０μＬをＴＣＡ沈殿で濃縮）
３：溶出液２ ４０μＬ相当
（ＰＢＳ＋１０ｍＭ ＥＤＴＡでの溶出画分 ｔｏｔａｌ ５０μＬのうち４０μＬをＴＣＡ沈殿で濃縮）
４：溶出後に得たＤｙｎａＢｅａｄｓ ５μＬ
（溶出後のＤｙｎａＢｅａｄｓ−ｍｏｕｓｅ ＩｇＧアプタマー複合体 ｔｏｔａｌ ５０μＬ）
５：抗ＦＬＡＧ抗体 １μＬ（１ｍｇ／ｍｌ １μｇ相当）
−：ＲＮＡなし
２８：配列番号２５に示すＲＮＡ
３１：配列番号２６に示すＲＮＡ
M: Marker (Precision Plus Protein Kaleidoscope Standards (manufactured by Bio-rad), catalog number: 161-0375)
1: Ascites-anti-FLAG antibody mixed solution 1 μL (11.5 μg protein + 1 μg anti-FLAG MAb equivalent)
2: Eluent 1 equivalent to 40 μL
(Eluted fraction with PBS alone total 40 μL of total 50 μL by TCA precipitation)
3: Eluent 2 equivalent to 40 μL
(Eluted fraction with PBS + 10 mM EDTA total 40 μL of total 50 μL by TCA precipitation)
4: DynaBeads 5 μL obtained after elution
(DynaBeads-mouse IgG aptamer complex after total 50 μL)
5: 1 μL of anti-FLAG antibody (equivalent to 1 μg of 1 mg / ml)
-: No RNA 28: RNA shown in SEQ ID NO: 25
31: RNA shown in SEQ ID NO: 26
（参考例１）
実施例８において、４００ｎｇの抗ＦＬＡＧマウスＩｇＧ抗体（標的物質２）に代えて、０．０１ｎｇ、０．０２５ｎｇ、０．０５ｎｇ、０．１ｎｇ、０．２５ｎｇ、０．５ｎｇ、１ｎｇ、２．５ｎｇ、５ｎｇ、１０ｎｇ、２５ｎｇ、５０ｎｇ及び１００ｎｇの抗体を用い、配列番号２４〜２７及び配列番号２８〜３１の配列からなるＲＮＡであって、９５度３分間の加熱後室温にて除冷しリフォールディングさせたものの代わりに、実施例１３で得た溶出液１、２、３、４及び５を１００倍及び１０００倍（溶出液３については、１０００倍及び１００００倍、溶出液５については、１００倍）に希釈して得た液を用いた以外は、実施例８と同様に行い、溶出液１、２、３、４及び５に含まれるＩｇＧの量を定量した。その結果を表１３に示す。
(Reference Example 1)
In Example 8, in place of 400 ng anti-FLAG mouse IgG antibody (target substance 2), 0.01 ng, 0.025 ng, 0.05 ng, 0.1 ng, 0.25 ng, 0.5 ng, 1 ng, 2.5 ng 5 ng, 10 ng, 25 ng, 50 ng and 100 ng antibodies, RNA consisting of the sequences of SEQ ID NO: 24-27 and SEQ ID NO: 28-31, which is heated at 95 ° C. for 3 minutes, cooled at room temperature, and refolded 100 times and 1000 times the eluates 1, 2, 3, 4 and 5 obtained in Example 13 (1000 times and 10000 times for eluate 3, and 100 times for eluate 5) The amount of IgG contained in the eluates 1, 2, 3, 4 and 5 was quantified in the same manner as in Example 8 except that the solution obtained by diluting was used. The results are shown in Table 13.
また、上記と同様に、実施例１３で得た溶出液２に代えて、実施例１４で得た溶出液１及び２を用いた以外は、上記と同様に、各溶出液に含まれるＩｇＧの量を定量した。その結果を表１３に示す。 Further, similarly to the above, except that the eluates 1 and 2 obtained in Example 14 were used in place of the eluate 2 obtained in Example 13, the IgG contained in each eluate was similar to the above. The amount was quantified. The results are shown in Table 13.
なお、ＩｇＧの量の定量には、上記の通りの抗体の希釈系列から導出した標準曲線に基づいて、定法に従い、行った。 The amount of IgG was determined according to a standard method based on a standard curve derived from the antibody dilution series as described above.
（実施例１５）
標的物質として、Ｎ−Ｔｅｒｍｉｎａｌ ＦＬＡＧ−ＢＡＰ Ｃｏｎｔｒｏｌ Ｐｒｏｔｅｉｎ（ＳＩＧＭＡ社製、カタログ番号；Ｐ７５８２）を用い、これをＳＤＳ−ＰＡＧＥ法に準じて、電気泳動を行い、得たバンドをＰＶＤＦ膜（商品名：Ｈｙｂｏｎｄ−Ｐ（ＧＥ社製）、カタログ番号：ＲＰＮ１４１６Ｆ）に転写した。これを、一次抗体として、標的物質２に示すＡＮＴＩ−ＦＬＡＧ Ｍ２ Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉｂｏｄｙ （ＳＩＧＭＡ社製、カタログ番号：Ｆ３１６５、ロット番号：０８６Ｋ６０１２）の５００倍希釈液に浸漬し、洗浄・ブロッキングを行った後、得た膜を、配列番号２４〜２７及び配列番号２８〜３１に示す配列のＲＮＡを有するＲＮＡ溶液（１００ｎＭ、１５μｇ／ｍＬ）で処理した。この膜を、化学発光基質としてＣｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ Ｐｅｒｏｘｉｄａｓｅ Ｓｕｂｓｔｒａｔｅ（ＳＩＧＭＡ社製、カタログ番号：ＣＰＳ−１−６０）を用いて発光させ、発光像を観察した。その結果を、図１２に示す。図１２において、各レーンは、下記に示す通りである。なお、発光時間は、２秒であった。また、図１２において、「ＳＡ−ＨＲＰ」は、上記のＲＮＡ溶液を用いずに発光させて得た発光像である。
(Example 15)
Using N-Terminal FLAG-BAP Control Protein (manufactured by SIGMA, catalog number; P7582) as a target substance, this was subjected to electrophoresis in accordance with the SDS-PAGE method, and the obtained band was PVDF membrane (trade name: It was transferred to Hybond-P (manufactured by GE), catalog number: RPN1416F). This was immersed in a 500-fold diluted solution of ANTI-FLAG M2 Monoclonal Antibody (manufactured by SIGMA, catalog number: F3165, lot number: 086K6012) shown as target substance 2 as a primary antibody, and washed and blocked. The obtained membrane was treated with an RNA solution (100 nM, 15 μg / mL) having RNAs of the sequences shown in SEQ ID NOs: 24-27 and 28-31. The film was allowed to emit light using Chemiluminescent Peroxidase Substrate (manufactured by SIGMA, catalog number: CPS-1-60) as a chemiluminescent substrate, and an emission image was observed. The result is shown in FIG. In FIG. 12, each lane is as shown below. The light emission time was 2 seconds. In FIG. 12, “SA-HRP” is a luminescence image obtained by emitting light without using the above RNA solution.
Ｍ：ビオチン化した分子量マーカー
１：１μｇのＦＬＡＧ−ＢＡＰ
２：２００ｎｇのＦＬＡＧ−ＢＡＰ
３：１００ｎｇのＦＬＡＧ−ＢＡＰ
M: Biotinylated molecular weight marker 1: 1 μg FLAG-BAP
2: 200 ng FLAG-BAP
3: 100 ng FLAG-BAP
（実施例１６）
実施例１５において、Ｎ−Ｔｅｒｍｉｎａｌ ＦＬＡＧ−ＢＡＰ Ｃｏｎｔｒｏｌ Ｐｒｏｔｅｉｎ（ＳＩＧＭＡ社製、カタログ番号；Ｐ７５８２）に加えて、標的物質２に示すＡＮＴＩ−ＦＬＡＧ Ｍ２ Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉｂｏｄｙ（ＳＩＧＭＡ社製、カタログ番号：Ｆ３１６５、ロット番号：０８６Ｋ６０１２）を用い、配列番号２４〜２７及び配列番号２８〜３１に示す配列のＲＮＡの代わりに、配列番号２５を用いた以外は、実施例１５と同様に行い、ＰＤＶＦ膜の発光像を得た。結果を図１３に示す。図１３において、各レーンは、下記の通りである。なお、発光時間は、８秒とした。
(Example 16)
In Example 15, in addition to N-Terminal FLAG-BAP Control Protein (manufactured by SIGMA, catalog number; P7582), ANTI-FLAG M2 Monoclonal Antibody (manufactured by SIGMA, catalog number: F3165, lot number) shown in target substance 2 : 086K6012), except that SEQ ID NO: 25 was used instead of the RNA of the sequences shown in SEQ ID NO: 24-27 and SEQ ID NO: 28-31, and a light emission image of the PDVF film was obtained. It was. The results are shown in FIG. In FIG. 13, each lane is as follows. The light emission time was 8 seconds.
Ｍ：ビオチン化した分子量マーカー
１：０．５μｇの抗ＦＬＡＧ−マウスＩｇＧ抗体
２：０．５μｇの抗ＦＬＡＧ−マウスＩｇＧ抗体及び１００ｎｇのＦＬＡＧ−ＢＡＰ
３：１００ｎｇのＦＬＡＧ−ＢＡＰ
M: Biotinylated molecular weight marker 1: 0.5 μg anti-FLAG-mouse IgG antibody 2: 0.5 μg anti-FLAG-mouse IgG antibody and 100 ng FLAG-BAP
3: 100 ng FLAG-BAP
以上、本発明の好適な実施の形態により本発明を説明した。ここでは特定の具体例を示して本発明を説明したが、特許請求の範囲に定義された本発明の広範な趣旨および範囲から逸脱することなく、これら具体例に様々な修正および変更を加えることができることは明らかである。すなわち、具体例の詳細および添付の図面により本発明が限定されるものと解釈してはならない。 The present invention has been described above by the preferred embodiments of the present invention. While the invention has been described with reference to specific embodiments, various modifications and changes may be made to the embodiments without departing from the broad spirit and scope of the invention as defined in the claims. Obviously you can. In other words, the present invention should not be construed as being limited by the details of the specific examples and the accompanying drawings.
Claims (32)
当該核酸分子は、該核酸分子の塩基配列をステム領域と一本鎖領域とに分割して得た二次構造候補のエネルギー関数を最小化することに基づく二次構造予測を用いて得た該核酸分子の二次構造において、ステム長さ３以上のステム領域を当該核酸分子の末端に有することを特徴とする核酸分子。 A nucleic acid molecule capable of binding to a mouse-derived IgG antibody,
The nucleic acid molecule is obtained using secondary structure prediction based on minimizing the energy function of a secondary structure candidate obtained by dividing the base sequence of the nucleic acid molecule into a stem region and a single-stranded region. A nucleic acid molecule having a stem region having a stem length of 3 or more at the end of the nucleic acid molecule in the secondary structure of the nucleic acid molecule.
請求項１乃至３１のいずれか一項に記載の核酸分子を含有する試薬を有することを特徴とする検出キット。 A detection kit for detecting binding to a mouse-derived IgG antibody,
32. A detection kit comprising a reagent containing the nucleic acid molecule according to any one of claims 1 to 31.
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|---|---|---|---|---|
| JP2016534723A (en) * | 2013-10-22 | 2016-11-10 | キム・ソンチョン | Marker for generating binding information between biomolecule and nucleic acid, method for producing the same, and biomolecule analysis method and apparatus using the same |
| JP2018027024A (en) * | 2016-08-15 | 2018-02-22 | 国立大学法人東京農工大学 | Detection method of aptamer and antibody |
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| JP2016534723A (en) * | 2013-10-22 | 2016-11-10 | キム・ソンチョン | Marker for generating binding information between biomolecule and nucleic acid, method for producing the same, and biomolecule analysis method and apparatus using the same |
| JP2018027024A (en) * | 2016-08-15 | 2018-02-22 | 国立大学法人東京農工大学 | Detection method of aptamer and antibody |
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