JP2014100095A - Fusion protein for protein detection and method for protein detection - Google Patents
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Abstract
Description
本発明は、タンパク質検出用融合タンパク質およびタンパク質の検出方法に関する。 The present invention relates to a fusion protein for protein detection and a protein detection method.
生体試料中には種々のタンパク質が存在するが、特定のタンパク質を検出、定量するための方法として、ＥＬＩＳＡ（Ｅｎｚｙｍｅ Ｌｉｎｋｅｄ ＩｍｍｕｎｏＳｏｒｂｅｎｔ Ａｓｓａｙ）等が知られている。 Various proteins exist in a biological sample. As a method for detecting and quantifying a specific protein, ELISA (Enzyme Linked Immunosorbent Assay) and the like are known.
ＥＬＩＳＡは、試料中に含まれる抗原等の特定のタンパク質を、酵素標識した抗体を用い、抗原抗体反応を利用して定量的に検出する方法であり、免疫検査等において汎用されている手法の１つである。ＥＬＩＳＡには、直接吸着法、サンドイッチ法、競合法等が知られている。 ELISA is a method for quantitatively detecting a specific protein such as an antigen contained in a sample using an enzyme-labeled antibody by using an antigen-antibody reaction, and is one of the techniques widely used in immunoassays and the like. One. For the ELISA, a direct adsorption method, a sandwich method, a competitive method, and the like are known.
例えば、固相の表面に吸着させた目的物質（抗原）に対する１次抗体を抗原抗体反応により結合させる。未反応の１次抗体を洗い流した後、酵素標識した標識２次抗体を添加して、再び抗原抗体反応により結合させる。ここで未反応の標識２次抗体を洗い流し、発色基質を添加すると、抗原の量に比例して発色反応が起こる。生成した発色物質の吸光度を吸光度計等により測定し、濃度既知の標準品を用いて作成した標準曲線から、抗原の量を定量することができる。 For example, a primary antibody against a target substance (antigen) adsorbed on the surface of a solid phase is bound by an antigen-antibody reaction. After washing away the unreacted primary antibody, an enzyme-labeled labeled secondary antibody is added, and again bound by antigen-antibody reaction. When unreacted labeled secondary antibody is washed away and a chromogenic substrate is added, a chromogenic reaction occurs in proportion to the amount of antigen. The absorbance of the generated chromogenic substance is measured with an absorptiometer or the like, and the amount of antigen can be quantified from a standard curve created using a standard product with a known concentration.
しかし、このような方法では、目的物質（抗原）に特異的に結合する１次抗体に対して特異的に結合する標識２次抗体が必要となり、複数種類の目的物質（抗原）を検出する場合、複数種類の１次抗体に対して、それぞれに特異的に結合する標識２次抗体を用意する必要があり、汎用性が低い。 However, such a method requires a labeled secondary antibody that specifically binds to the primary antibody that specifically binds to the target substance (antigen), and detects multiple types of target substances (antigens). In addition, it is necessary to prepare labeled secondary antibodies that specifically bind to each of a plurality of types of primary antibodies, and the versatility is low.
一方、標識２次抗体の代わりに、アルカリホスファターゼ等の酵素とプロテインＧとを化学反応で結合した酵素標識プロテインＧを用いる方法がある。プロテインＧは、連鎖球菌の細胞壁由来タンパク質で、ほとんどの哺乳動物のＩｇＧと結合する性質を持つ。このような酵素標識プロテインＧを用いれば多くの１次抗体と結合可能であり、複数種類の目的物質（抗原）を検出する場合でも、それぞれに特異的に結合する抗体を用意しなくてもよく、汎用性が高い。 On the other hand, instead of the labeled secondary antibody, there is a method using enzyme-labeled protein G obtained by binding an enzyme such as alkaline phosphatase and protein G by a chemical reaction. Protein G is a streptococcal cell wall-derived protein that binds to most mammalian IgG. When such enzyme-labeled protein G is used, it can bind to many primary antibodies, and even when detecting multiple types of target substances (antigens), it is not necessary to prepare antibodies that specifically bind to each of them. High versatility.
しかし、アルカリホスファターゼ等の酵素で標識した酵素標識プロテインＧを用いる方法は、検出感度が低いという問題があった。また、熱安定性が低いものもあり、取扱中に失活する場合があった。 However, the method using enzyme-labeled protein G labeled with an enzyme such as alkaline phosphatase has a problem of low detection sensitivity. In addition, some have low thermal stability and may be deactivated during handling.
本発明は、汎用性が高く、検出感度が高く、かつ安定性の高いタンパク質検出用融合タンパク質およびそれを用いたタンパク質の検出方法を提供することにある。 An object of the present invention is to provide a protein detection fusion protein having high versatility, high detection sensitivity, and high stability, and a protein detection method using the same.
本発明は、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインのうち少なくとも１つを含むタンパク質ドメインと、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼとを融合したものであるタンパク質検出用融合タンパク質である。 The present invention provides a protein domain comprising at least one of the C2 domain of protein G and the C3 domain of protein G, and a fused alkaline phosphatase in which partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase are fused. It is a fusion protein for protein detection that is fused.
また、前記タンパク質検出用融合タンパク質において、前記タンパク質ドメインが、プロテインＡのＢドメイン、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインが結合されたものであることが好ましい。 In the protein detection fusion protein, the protein domain is preferably one in which the B domain of protein A, the C2 domain of protein G, and the C3 domain of protein G are bound.
また、前記タンパク質検出用融合タンパク質において、前記融合型アルカリホスファターゼが、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼＩＰＰまたはＩＩＰであることが好ましい。 In the fusion protein for protein detection, the fused alkaline phosphatase is preferably fused alkaline phosphatase IPP or IIP in which partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase are fused.
また、本発明は、タンパク質の検出方法であって、前記タンパク質検出用融合タンパク質と、対象物中に存在するタンパク質とを直接または間接的に結合させ、結合している前記タンパク質検出用融合タンパク質のアルカリホスファターゼ部分を標識部分として検出するタンパク質の検出方法である。 The present invention also relates to a method for detecting a protein, wherein the fusion protein for protein detection is directly or indirectly bound to a protein present in an object, and the fusion protein for protein detection is bound. This is a protein detection method in which an alkaline phosphatase moiety is detected as a label moiety.
本発明では、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインのうち少なくとも１つを含むタンパク質ドメインと、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼとを融合させることにより、汎用性が高く、検出感度が高く、かつ安定性の高いタンパク質検出用融合タンパク質およびそれを用いたタンパク質の検出方法を提供することができる。 In the present invention, a protein domain comprising at least one of the C2 domain of protein G and the C3 domain of protein G, and a fused alkaline phosphatase in which partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase are fused. By fusing, it is possible to provide a protein detection fusion protein with high versatility, high detection sensitivity, and high stability, and a protein detection method using the same.
本発明の実施の形態について以下説明する。本実施形態は本発明を実施する一例であって、本発明は本実施形態に限定されるものではない。 Embodiments of the present invention will be described below. This embodiment is an example for carrying out the present invention, and the present invention is not limited to this embodiment.
本実施形態に係るタンパク質検出用融合タンパク質は、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインのうち少なくとも１つを含むタンパク質ドメインと、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼとを融合したものである。 The fusion protein for protein detection according to this embodiment comprises a protein domain containing at least one of the C2 domain of protein G and the C3 domain of protein G, and partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase. It is a fusion of fused alkaline phosphatase.
本発明者らは、検出対象であるタンパク質と直接または間接的に結合するタンパク質ドメインとして、プロテインＧに着目した。プロテインＧは、連鎖球菌の細胞壁由来タンパク質で、ほとんどの哺乳動物のＩｇＧと結合する性質を持つ。このプロテインＧをアルカリホスファターゼ標識し、標識２次抗体等に代わるものとして利用できないかを検討した。 The present inventors have focused on protein G as a protein domain that directly or indirectly binds to a protein to be detected. Protein G is a streptococcal cell wall-derived protein that binds to most mammalian IgG. This protein G was labeled with alkaline phosphatase, and it was examined whether it could be used as a substitute for a labeled secondary antibody.
また、本発明者らは、検出感度および安定性に寄与する酵素標識としてのアルカリホスファターゼのうち、ヒト胎盤型アルカリホスファターゼ（ＰＬＡＰ）は熱安定性が高く、ヒト小腸型アルカリホスファターゼ（ＩＡＰ）は比活性が高いこと、そのヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼ両者の配列をキメラ化することで、高活性かつ安定性の高いキメラタンパク質が創出されることに着目した。なお、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼはアミノ酸配列にして８８％の相同性を持つ。 In addition, among the alkaline phosphatases serving as enzyme labels that contribute to detection sensitivity and stability, the present inventors show that human placental alkaline phosphatase (PLAP) has high thermal stability, and human small intestinal alkaline phosphatase (IAP) We focused on the fact that a highly active and highly stable chimeric protein was created by chimerizing both human placental alkaline phosphatase and human small intestine alkaline phosphatase sequences. Human placental alkaline phosphatase and human small intestine alkaline phosphatase have 88% homology in amino acid sequence.
そこで、鋭意検討したところ、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインのうち少なくとも１つを含むタンパク質ドメインと、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼとを融合させることにより、汎用性が高く、検出感度が高く、かつ安定性の高いタンパク質検出用融合タンパク質が得られることを見出した。 As a result of intensive studies, a fusion type alkali comprising a protein domain containing at least one of the C2 domain of protein G and the C3 domain of protein G, and partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase, respectively. It was found that a fusion protein for protein detection having high versatility, high detection sensitivity, and high stability can be obtained by fusing with phosphatase.
タンパク質ドメインとしては、プロテインＧのＩｇＧ結合ドメインであるＣ２ドメインおよびＣ３ドメインのうち少なくとも１つを含むものであればよく、特に制限はないが、抗体から脱離しにくい等の点から、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインが連結されているものを含むことが好ましい。また、プロテインＧの抗体等に対する反応性が向上する等の点から、プロテインＡのＢドメインを含むことが好ましい。特に、プロテインＡのＢドメイン、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインが結合されたものであることが好ましい。 The protein domain is not particularly limited as long as it includes at least one of the C2 domain and C3 domain that are IgG binding domains of protein G. It is preferable to include those in which the C2 domain and the C3 domain of protein G are linked. Moreover, it is preferable that the B domain of protein A is included from the point that the reactivity with respect to the antibody etc. of protein G improves. In particular, the B domain of protein A, the C2 domain of protein G, and the C3 domain of protein G are preferably combined.
ヒト胎盤型アルカリホスファターゼ（ＰＬＡＰ）とヒト小腸型アルカリホスファターゼ（ＩＡＰ）それぞれの部分配列を融合した融合型アルカリホスファターゼとしては、比活性が高く、熱安定性が高い等の点から、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼＩＰＰまたはＩＩＰ（非特許文献３参照）が好ましく、生産性や、比活性が高く、熱安定性が高い等の点から、ＩＰＰが好ましい。 As a fusion type alkaline phosphatase in which partial sequences of human placenta type alkaline phosphatase (PLAP) and human small intestine type alkaline phosphatase (IAP) are fused, human placental type alkali is used because of its high specific activity and high thermal stability. Fusion type alkaline phosphatase IPP or IIP (see Non-Patent Document 3) in which partial sequences of phosphatase and human small intestine type alkaline phosphatase are fused is preferable. From the viewpoint of productivity, high specific activity, and high thermal stability, etc. IPP is preferred.
融合型アルカリホスファターゼのＣ末端側にプロテインＧのＣ２ドメインを融合させたものでもよいし、Ｎ末端側に融合させたものでもよいが、生産性等の点から、融合型アルカリホスファターゼのＣ末端側にプロテインＧのＣ２ドメインを融合させたものであることが好ましい。 The C-terminal side of protein G may be fused to the C-terminal side of the fused alkaline phosphatase, or may be fused to the N-terminal side. From the viewpoint of productivity, the C-terminal side of the fused alkaline phosphatase It is preferable that the C2 domain of protein G is fused with the G2.
本実施形態に係るタンパク質検出用融合タンパク質は、６個程度の連続するヒスチジン（Ｈｉｓ）残基からなるタグペプチドの一種であるＨｉｓタグ等のタグを有していてもよい。 The fusion protein for protein detection according to the present embodiment may have a tag such as a His tag which is a kind of tag peptide composed of about 6 consecutive histidine (His) residues.
図１に、本実施形態に係るタンパク質検出用融合タンパク質の一例の構造を模式的に示す。本実施形態に係るタンパク質検出用融合タンパク質は、例えば、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼ（キメラＡＰ）のＣ末端側に、プロテインＡのＢドメイン、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインが結合され、プロテインＧドメインのＣ末端側にＨｉｓタグが付加されたものである。 FIG. 1 schematically shows an example of the structure of a protein detection fusion protein according to this embodiment. The protein detection fusion protein according to the present embodiment includes, for example, protein A B on the C-terminal side of fusion alkaline phosphatase (chimeric AP) in which partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase are fused. The domain, the C2 domain of protein G, and the C3 domain of protein G are combined, and a His tag is added to the C-terminal side of the protein G domain.
本実施形態に係るタンパク質検出用融合タンパク質は、遺伝子工学的手法により、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインのうち少なくとも１つを含むタンパク質ドメインと、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼとを融合した融合タンパク質として発現させて得ることができる。また、その際、プロテインＧドメインのＣ末端側にＨｉｓタグ等のタグを付与してもよい。 The protein detection fusion protein according to the present embodiment comprises a protein domain containing at least one of the C2 domain of protein G and the C3 domain of protein G, human placental alkaline phosphatase, and human small intestine alkaline by genetic engineering techniques. It can be obtained by expressing as a fusion protein fused with a fusion alkaline phosphatase fused with a partial sequence of each phosphatase. At that time, a tag such as a His tag may be added to the C-terminal side of the protein G domain.
融合タンパク質の精製は、Ｎ末端またはＣ末端に付加した精製用ペプチドタグ（例えば、（Ｈｉｓ）６−ｔａｇ（ヘキサヒスチジンタグ））を利用し、ゲル濾過クロマトグラフィ、固定化金属イオンアフィニティクロマトグラフィ等により行うことができる。 Purification of the fusion protein is performed by gel filtration chromatography, immobilized metal ion affinity chromatography, etc. using a peptide tag for purification (for example, (His) 6-tag (hexahistidine tag)) added to the N-terminus or C-terminus. be able to.
融合タンパク質のアミノ酸配列の確認は当該タンパク質をコードするプラスミドベクターの遺伝子配列をＤＮＡシーケンサにて確認することができる。融合タンパク質の精製の確認は、ＳＤＳ−ＰＡＧＥ等で行うことができる。 The amino acid sequence of the fusion protein can be confirmed using a DNA sequencer for the gene sequence of the plasmid vector encoding the protein. Confirmation of the purification of the fusion protein can be performed by SDS-PAGE or the like.
本実施形態に係るタンパク質検出用融合タンパク質によれば、ほぼ全ての動物種の１次抗体を検出可能である。本実施形態に係るタンパク質検出用融合タンパク質の検出感度は、従来の標識プロテインＧより高く、安定性も高い。また、従来の標識２次抗体と検出感度は同等以上であり、汎用性が高い。本実施形態に係るタンパク質検出用融合タンパク質を用いれば多くの１次抗体と結合可能であり、複数種類の目的物質（抗原）を検出する場合でも、それぞれに特異的に結合する抗体を用意しなくてもよく、汎用性が高い。 According to the fusion protein for protein detection according to the present embodiment, primary antibodies of almost all animal species can be detected. The detection sensitivity of the fusion protein for protein detection according to this embodiment is higher than that of the conventional labeled protein G, and also has high stability. Further, the detection sensitivity is equal to or higher than that of the conventional labeled secondary antibody, and the versatility is high. If the fusion protein for protein detection according to this embodiment is used, it can bind to many primary antibodies, and even when detecting multiple types of target substances (antigens), it is not necessary to prepare antibodies that specifically bind to each of them. It is good and versatility is high.
また、本実施形態に係るタンパク質検出用融合タンパク質は、変性ＩｇＧを検出しない傾向にある。そのため、例えば、ＩｇＧが多く含まれるサンプルであっても、内在性ＩｇＧが検出されない（後述する実施例４参照）。また、免疫沈降のキャプチャー抗体が検出されない（後述する実施例３参照）という利点がある。 Moreover, the fusion protein for protein detection according to the present embodiment tends not to detect denatured IgG. Therefore, for example, even in a sample containing a large amount of IgG, endogenous IgG is not detected (see Example 4 described later). In addition, there is an advantage that an immunoprecipitation capture antibody is not detected (see Example 3 described later).
＜タンパク質の検出方法＞
本実施形態に係るタンパク質の検出方法は、前記タンパク質検出用融合タンパク質と、対象物中に存在するタンパク質とを直接または間接的に結合させ、結合しているタンパク質検出用融合タンパク質のアルカリホスファターゼ部分を標識部分として検出する方法である。
<Protein detection method>
In the protein detection method according to the present embodiment, the protein detection fusion protein and the protein present in the target are directly or indirectly bound, and the alkaline phosphatase portion of the bound protein detection fusion protein is bound. It is a method of detecting as a labeling moiety.
本実施形態に係るタンパク質検出用融合タンパク質およびタンパク質の検出方法は、例えば、
ウエスタンブロット、ＥＬＩＳＡ、
免疫組織化学（免疫染色）等の基礎研究分野や、
病理検査分野等に利用することができる。
The protein detection fusion protein and protein detection method according to this embodiment include, for example,
Western blot, ELISA,
Basic research fields such as immunohistochemistry (immunostaining)
It can be used in the pathological examination field.
以下、実施例および比較例を挙げ、本発明をより具体的に詳細に説明するが、本発明は、以下の実施例に限定されるものではない。なお、下記実施例中、室温とは２０〜２５℃である。 EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated in detail more concretely, this invention is not limited to a following example. In the following examples, room temperature is 20 to 25 ° C.
＜実施例１＞
融合型アルカリホスファターゼ（ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合タンパク質ＩＰＰ）と、プロテインＡのＢドメイン、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメイン（ＰＧ１（ｐＡ−ｐＧ−ｐＧ））と、Ｈｉｓタグとを接続した融合タンパク質遺伝子配列を、ｐｃＤＮＡ３．１Ｖ−５ ＨｉｓＡベクター（ｉｎｖｉｔｒｏｇｅｎ）に導入して作製した発現ベクター（ｐＰＧ１−ＩＰＰ＃１（配列番号：１））、および、その融合タンパク質遺伝子配列のＧＣ含量を５４％程度に下げると共に回文配列の除去を行った配列を、ｐｃＤＮＡ３．１Ｖ−５ ＨｉｓＡベクターに導入して作製した発現ベクター２種（以下、ｐＰＧ１−ＩＰＰ＃２（配列番号：２）およびｐＩＰＰ−ＰＧ１＃２（配列番号：３））の、ヒト胎児腎由来細胞株ＨＥＫ２９３Ｔにおける一過性発現、および発現タンパク質の金属固定化アフィニティカラム（ＩＭＡＣカラム、ＴＡＬＯＮ）による精製を行った。以下、これら３種の発現ベクターによる生産物をそれぞれＰＧ１−ＩＰＰ＃１（配列番号：４）、ＰＧ１−ＩＰＰ＃２（配列番号：５）およびＩＰＰ−ＰＧ１＃２（配列番号：６）と略記する。
<Example 1>
Fusion alkaline phosphatase (fusion protein IPP in which partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase are fused), protein A B domain, protein G C2 domain and protein G C3 domain (PG1 (pA -PG-pG))) and an expression vector (pPG1-IPP # 1 (SEQ ID NO: 1)) prepared by introducing a fusion protein gene sequence connecting a His tag into a pcDNA3.1V-5 HisA vector (invitrogen) ), And two expression vectors (hereinafter referred to as the following) prepared by introducing a sequence obtained by reducing the GC content of the fusion protein gene sequence to about 54% and removing the palindromic sequence into the pcDNA3.1V-5 HisA vector. pPG1-IPP # 2 (SEQ ID NO: 2) and pIPP-PG1 # 2 (SEQ ID NO: 3)) were transiently expressed in the human fetal kidney-derived cell line HEK293T, and the expressed protein was purified by a metal-immobilized affinity column (IMAC column, TALON). . Hereinafter, the products of these three expression vectors are abbreviated as PG1-IPP # 1 (SEQ ID NO: 4), PG1-IPP # 2 (SEQ ID NO: 5) and IPP-PG1 # 2 (SEQ ID NO: 6), respectively. To do.
［材料と試験方法］
１．発現プラスミド
使用したプラスミド
ａ．ｐＩＲＥＳｎｅｏ３（コントロールベクター）
ｂ．ｐＰＧ１−ＩＰＰ＃１（ｐＧ−ｐＧ−ｐＡ−ＩＰＰ）
ｃ．ｐＰＧ１−ＩＰＰ＃２（ｐＧ−ｐＧ−ｐＡ−ＩＰＰ）
ｄ．ｐＩＰＰ−ＰＧ１＃２（ＩＰＰ−ｐＡ−ｐＧ−ｐＧ）
[Materials and test methods]
1. Expression plasmids Plasmids used a. pIRESneo3 (control vector)
b. pPG1-IPP # 1 (pG-pG-pA-IPP)
c. pPG1-IPP # 2 (pG-pG-pA-IPP)
d. pIPP-PG1 # 2 (IPP-pA-pG-pG)
２．培養細胞
ヒト胎児腎細胞由来でＳＶ４０ｌａｒｇｅＴ抗原を発現するＨＥＫ２９３Ｔ細胞は、理研細胞バンクより入手した（＃ＲＣＢ２２０２）。
2. Cultured cells HEK293T cells derived from human embryonic kidney cells and expressing SV40 large T antigen were obtained from RIKEN Cell Bank (# RCB2202).
３．一過性分泌発現試験
（１）細胞培養
ＨＥＫ２９３Ｔ細胞の培養に使用する増殖培地には、１０％ＦＢＳと２ｍＭ Ｌ−ｇｌｕｔａｍｉｎｅを含むＤＭＥＭ培地を用いた。ＨＥＫ２９３Ｔ細胞は、１０ｃｍ−ｄｉｓｈ中、ＣＯ２インキュベータ（３７℃、５％ＣＯ２）で静置培養し、７０〜８０％コンフルエントの細胞集団を３日に１度、１／８に希釈して継代した。トランスフェクション用の細胞は、１０ｃｍ−ｄｉｓｈに培養した８０％コンフルエントのＨＥＫ２９３Ｔ細胞を、トランスフェクションを行う２４時間前に１／２に希釈継代し作製した。
3. Transient secretion expression test (1) Cell culture A DMEM medium containing 10% FBS and 2 mM L-glutamine was used as a growth medium used for culturing HEK293T cells. HEK293T cells were statically cultured in a 10 cm-dish in a CO 2 incubator (37 ° C., 5% CO 2 ), and a 70-80% confluent cell population was diluted 1/8 once every 3 days and subcultured. I made it. Cells for transfection were prepared by subcultured 80% confluent HEK293T cells cultured at 10 cm-dish to 1/2 before 24 hours of transfection.
（２）トランスフェクション
ＨＥＫ２９３Ｔ細胞へのトランスフェクションは、Ｌｉｐｏｆｅｃｔａｍｉｎｅ ＬＴＸ Ｐｌｕｓ試薬（ｉｎｖｉｔｒｏｇｅｎ、＃１５３３８−１００）を使用した。また、トランスフェクションに使用するプラスミドは、以下（３）で示すものである。
(2) Transfection Lipofectamine LTX Plus reagent (Invitrogen, # 15338-100) was used for transfection into HEK293T cells. Moreover, the plasmid used for transfection is shown by the following (3).
トランスフェクションに先立ち、１０ｃｍ−ｄｉｓｈ×４枚のＨＥＫ２９３Ｔ細胞（８０％コンフルエント）を準備し、培養液を除き、１０ｃｍ−ｄｉｓｈ １枚あたり５ｍＬのＯｐｔｉ−ＭＥＭ Ｉ Ｒｅｄｕｃｅｄ−Ｓｅｒｕｍ Ｍｅｄｉｕｍ（ｉｎｖｉｔｒｏｇｅｎ、＃３１９８５−０７０）で洗浄し、１０ｃｍ−ｄｉｓｈ １枚あたり９ｍＬのＯｐｔｉ−ＭＥＭ培地を添加した。 Prior to transfection, 10 cm-dish × 4 HEK293T cells (80% confluent) were prepared, the culture medium was removed, and 5 mL of Opti-MEM I Reduced-Serum Medium (invitrogen, # 31985) per 10 cm-dish. 070) and 9 mL of Opti-MEM medium was added per 10 cm-dish.
続いて以下の表２に示す用量および時間でＯｐｔｉ−ＭＥＭ培地と発現プラスミドＤＮＡとトランスフェクション試薬（Ｐｌｕｓ試薬およびＬＴＸ試薬）を混合し、室温で放置してトランスフェクション混合液を作製した。 Subsequently, Opti-MEM medium, expression plasmid DNA, and transfection reagent (Plus reagent and LTX reagent) were mixed at the doses and times shown in Table 2 below, and allowed to stand at room temperature to prepare a transfection mixture.
各トランスフェクション混合液は、８ｍＬのＯｐｔｉ−ＭＥＭ培地を加えた１０ｃｍ−ｄｉｓｈ上のＨＥＫ２９３Ｔ細胞に滴下し、７２時間培養を継続して一過性発現を行った。 Each transfection mixture was added dropwise to HEK293T cells on 10 cm-dish to which 8 mL of Opti-MEM medium was added, and culture was continued for 72 hours for transient expression.
（３）調製したエンドトキシンフリープラスミド
調製したエンドトキシンフリープラスミドを表１に示す。
(3) Prepared endotoxin-free plasmid Table 1 shows the prepared endotoxin-free plasmid.
（４）トランスフェクション混合液の調製
トランスフェクション混合液の調製方法を表２に示す。
(4) Preparation of transfection mixture The preparation method of the transfection mixture is shown in Table 2.
（５）培養上清の回収
トランスフェクション７２時間後の培養上清を回収し、１，５００ｒｐｍ、４℃で１０分間遠心分離を行い、Ｓｙｒｉｎｇｅ−Ｆｉｌｔｅｒ０．２２μm（ＴＰＰ、＃９９７２２）でろ過し、ろ液を４℃で保存した。各培養上清のろ液１０ｍＬのうち、１ｍＬはサンプルとし、１ｍＬを培養上清の発現確認のために、残り８ｍＬをＨｉｓタグによる簡易カラム精製のためにそれぞれ使用した。
(5) Collection of culture supernatant The culture supernatant 72 hours after transfection was collected, centrifuged at 1,500 rpm, 4 ° C. for 10 minutes, filtered through Syringe-Filter 0.22 μm (TPP, # 99722), The filtrate was stored at 4 ° C. Of the 10 mL of the filtrate of each culture supernatant, 1 mL was used as a sample, 1 mL was used for confirming the expression of the culture supernatant, and the remaining 8 mL was used for simple column purification using a His tag.
４．培養上清の発現確認と精製
（１）培養上清の発現確認
フィルタろ過済みの培養上清各１ｍＬ分をＡｍｉｃｏｎ Ｕｌｔｒａ−０．５ Ｃｅｎｔｒｉｆｕｇａｌ Ｆｉｌｔｅｒ Ｕｎｉｔ ｗｉｔｈ Ｕｌｔｒａｃｅｌ−１０ｍｅｍｂｒａｎｅ（１０，０００ＭＷＣＯ、Ｍｉｌｌｉｐｏｒｅ、＃ＵＦＣ５０１０９６）で５０μＬに濃縮（２０倍濃縮）した。濃縮した各培養上清１０μＬ分（原液２００μＬ分に相当）をＳＤＳ−ＰＡＧＥにより分離し、ＣＢＢ染色および抗Ｐｅｎｔａ−Ｈｉｓ抗体（Ｐｅｎｔａ−Ｈｉｓ Ａｎｔｉｂｏｄｙ、Ｑｉａｇｅｎ、＃３４６６０）を使用したイムノブロッティングにより、分泌発現の有無を確認した。
4). Confirmation and Purification of Culture Supernatant (1) Confirmation of Culture Supernatant Expression 1 mL each of the filtered culture supernatant was added to Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-10 membrane (10,000 MWCO, Millipore, # UFC501096) ) To 50 μL (20-fold concentration). 10 μL of each concentrated culture supernatant (corresponding to 200 μL of stock solution) was separated by SDS-PAGE and secreted by CBB staining and immunoblotting using an anti-Penta-His antibody (Penta-His Antibody, Qiagen, # 34660). The presence or absence of expression was confirmed.
（２）Ｈｉｓタグによる簡易カラム精製
Ｈｉｓタグによるカラム精製には、ＴＡＬＯＮ Ｍｅｔａｌ Ａｆｆｉｎｉｔｙ Ｒｅｓｉｎ（Ｃｌｏｎｔｅｃｈ、＃６３５５０４）を使用した。フィルタろ過済みの培養上清各８ｍＬ分をＡｍｉｃｏｎ Ｕｌｔｒａ−４ Ｃｅｎｔｒｉｆｕｇａｌ Ｆｉｌｔｅｒ Ｕｎｉｔ ｗｉｔｈ Ｕｌｔｒａｃｅｌ−１０ｍｅｍｂｒａｎｅ（１０，０００ＭＷＣＯ、Ｍｉｌｌｉｐｏｒｅ、＃ＵＦＣ８０１０２４）を使用して５００μＬに濃縮（１６倍濃縮）し、５ｍＬの結合／洗浄バッファ（５０ｍＭ Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．４）、３００ｍＭ ＮａＣｌ、１０ｍＭ ｉｍｉｄａｚｏｌｅ）を加えて希釈し、カラムへのアプライサンプルとした。
(2) Simple column purification with His tag For column purification with His tag, TALON Metal Affinity Resin (Clontech, # 635504) was used. Concentrate 8 mL each of the filtered culture supernatant to 500 μL using Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane (10,000 MWCO, Millipore, # UFC801024), 5 mL binding / Washing buffer (50 mM Na-phosphate (pH 7.4), 300 mM NaCl, 10 mM imidazole) was added and diluted to obtain an applied sample to the column.
各アプライサンプルは、結合／洗浄バッファで平衡化したＴＡＬＯＮカラム（ｂｅｄ ｖｏｌ．２００μＬ）にアプライし、その後それぞれのカラムを２ｍＬの結合／洗浄バッファで２回洗浄した。溶出は段階溶出で４００μＬずつ３回施行した。１回目と２回目は、４００μＬの溶出バッファ＃１５０（５０ｍＭ Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．４）、３００ｍＭ ＮａＣｌ、１５０ｍＭ ｉｍｉｄａｚｏｌｅ）で溶出した（以下溶出＃１、溶出＃２）。３回目は、４００μＬの溶出バッファ＃５００（５０ｍＭ Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．４）、３００ｍＭ ＮａＣｌ、５００ｍＭ ｉｍｉｄａｚｏｌｅ）で溶出した（以下溶出＃３）。 Each apply sample was applied to a TALON column (bed vol. 200 μL) equilibrated with binding / wash buffer, and then each column was washed twice with 2 mL binding / wash buffer. Elution was carried out 3 times with 400 μL in step elution. In the first and second rounds, elution was carried out with 400 μL of elution buffer # 150 (50 mM Na-phosphate (pH 7.4), 300 mM NaCl, 150 mM imidazole) (hereinafter, elution # 1, elution # 2). The third elution was performed with 400 μL of elution buffer # 500 (50 mM Na-phosphate (pH 7.4), 300 mM NaCl, 500 mM imidazole) (hereinafter, elution # 3).
各精製画分（アプライサンプル、素通り画分、および溶出＃１、溶出＃２、溶出＃３）それぞれ１０μＬ分を、それぞれＳＤＳ−ＰＡＧＥにより分離し、ＣＢＢ染色およびＰｅｎｔａ−Ｈｉｓ抗体を使用したイムノブロッティングにより確認した。 10 μL of each purified fraction (apply sample, flow-through fraction, and elution # 1, elution # 2, elution # 3) was separated by SDS-PAGE, and immunoblotting using CBB staining and Penta-His antibody. Confirmed by
（３）ＳＤＳ−ＰＡＧＥとイムノブロティング
ＳＤＳ−ＰＡＧＥには、ｅ−ＰＡＧＥＬ １０％（ＡＴＴＯ、＃Ｅ−Ｒ１０Ｌ）を使用した。分子量マーカは、Ｐｒｅｓｔａｉｎｅｄ Ｐｒｏｔｅｉｎ Ｍａｒｋｅｒ，Ｂｒｏａｄ Ｒａｎｇｅ（ＮＥＢ、＃Ｐ７７０８Ｓ）を使用した。また、ＳＤＳ−ＰＡＧＥゲルからの転写には、Ｈｙｂｏｎｄ−ＥＣＬ（ＧＥ、＃ＲＰＮ３０３２Ｄ）を使用した。
(3) SDS-PAGE and immunoblotting e-PAGEEL 10% (ATTO, # E-R10L) was used for SDS-PAGE. As the molecular weight marker, Prestained Protein Marker, Broad Range (NEB, # P7708S) was used. In addition, Hybond-ECL (GE, # RPN3032D) was used for transfer from the SDS-PAGE gel.
抗Ｈｉｓ抗体は、マウスモノクローナル抗体（Ｐｅｎｔａ−Ｈｉｓ Ａｎｔｉｂｏｄｙ、Ｑｉａｇｅｎ、＃３４６６０）を最終濃度０．４μｇ／ｍＬで使用した。また、二次抗体は、ＩＲＤｙｅ８００ＣＷ 標識ヤギ抗マウス抗体（＃６１０−７３１−１２４、Ｒｏｃｋｌａｎｄ）を最終濃度２μg／ｍＬで使用した。 As the anti-His antibody, a mouse monoclonal antibody (Penta-His Antibody, Qiagen, # 34660) was used at a final concentration of 0.4 μg / mL. As the secondary antibody, IRDye800CW labeled goat anti-mouse antibody (# 610-731-124, Rockland) was used at a final concentration of 2 μg / mL.
［結果］
（１）培養上清の発現確認
一過性分泌発現させた各培養上清（各レーン２００μＬ分）のＣＢＢ染色と抗Ｐｅｎｔａ−Ｈｉｓ抗体によるイムノブロットの結果を図２に示す。ＳＤＳ−ＰＡＧＥのＣＢＢ染色および抗Ｐｅｎｔａ−Ｈｉｓ抗体によるイムノブロットのいずれにおいても、ｐＰＧ１−ＩＰＰ＃１による一過性発現では微弱にしか検出できなかった標的タンパク質を、ｐＩＰＰ−ＰＧ１＃２およびｐＰＧ１−ＩＰＰ＃２による一過性発現では、８５ｋＤａ付近および１７０ｋＤａ付近に、顕著なバンドとして確認できた。
[result]
(1) Confirmation of expression of culture supernatant FIG. 2 shows the results of immunoblotting with CBB staining and anti-Penta-His antibody of each culture supernatant (200 μL for each lane) that was transiently expressed. In both SDS-PAGE CBB staining and immunoblotting with anti-Penta-His antibody, target proteins that could only be weakly detected by transient expression with pPG1-IPP # 1 were detected as pIPP-PG1 # 2 and pPG1- In transient expression by IPP # 2, it was confirmed as a prominent band around 85 kDa and around 170 kDa.
ｐＩＰＰ−ＰＧ１＃２とｐＰＧ１−ＩＰＰ＃２について比較すると、ＳＤＳ−ＰＡＧＥのＣＢＢ染色においても、抗Ｐｅｎｔａ−Ｈｉｓ抗体によるイムノブロットにおいても、ｐＩＰＰ−ＰＧ１＃２の方が圧倒的に強い発現となった。 Comparing pIPP-PG1 # 2 and pPG1-IPP # 2, pIPP-PG1 # 2 was overwhelmingly expressed in both SDS-PAGE CBB staining and immunoblotting with anti-Penta-His antibody. It was.
ここでは標準アルブミン溶液を用いた定量は行わなかったが、バンドの濃度からの概算として、ｐＩＰＰ−ＰＧ１＃２とｐＰＧ１−ＩＰＰ＃２はそれぞれ１０μg／ｍＬ程度の発現レベルとなったことが推定される。 Although quantification using a standard albumin solution was not performed here, it was estimated that pIPP-PG1 # 2 and pPG1-IPP # 2 each had an expression level of about 10 μg / mL as an estimate from the band concentration. The
（２）Ｈｉｓタグによる簡易カラム精製
各精製フラクション（各レーン１０μＬ分）のＣＢＢ染色と抗Ｐｅｎｔａ−Ｈｉｓ抗体によるイムノブロットの結果を図３に示す。ＳＤＳ−ＰＡＧＥのＣＢＢ染色および抗Ｐｅｎｔａ−Ｈｉｓ抗体によるイムノブロットの結果、結合／洗浄バッファに平衡化後のアプライサンプルからは、上記（１）のろ過培養上清と同様に、８５ｋＤａおよび１７０ｋＤａ付近に二つの顕著なバンドが検出される一方、ＴＡＬＯＮカラムによるＨｉｓタグ精製では、目的物の大部分はアプライサンプルと同様な８５ｋＤａ付近および１７０ｋＤａ付近の２つのバンドとして溶出＃１の画分に、また一部はフロースルーと溶出＃２の画分に検出された。溶出＃３画分での検出は、わずかであった。
(2) Simple column purification with His tag FIG. 3 shows the results of CBB staining of each purified fraction (10 μL for each lane) and immunoblotting with anti-Penta-His antibody. As a result of SDS-PAGE CBB staining and immunoblotting with anti-Penta-His antibody, the applied sample after equilibration to the binding / washing buffer was found to be around 85 kDa and 170 kDa, similar to the filtration culture supernatant of (1) above. While two prominent bands are detected, in His tag purification using a TALON column, most of the target substance is in the fraction of elution # 1 as two bands around 85 kDa and 170 kDa, similar to the applied sample, and one more Parts were detected in the flow-through and elution # 2 fractions. Detection in the elution # 3 fraction was negligible.
結論として、培養上清の発現産物は、結合／洗浄バッファ（５０ｍＭ Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．４）、３００ｍＭ ＮａＣｌ、１０ｍＭ ｉｍｉｄａｚｏｌｅ）の平衡化条件で結合し、溶出バッファ１５０（５０ｍＭ Ｎａ−ｐｈｏｓｐｈａｔｅ（ｐＨ７．４）、３００ｍＭ ＮａＣｌ、１５０ｍＭ ｉｍｉｄａｚｏｌｅ）の溶出条件で溶出されることが判明した。このことから、培養上清からの目的産物のＨｉｓタグによる簡易カラム精製は可能と結論される。 In conclusion, the expression product of the culture supernatant is bound under the equilibration conditions of binding / washing buffer (50 mM Na-phosphate (pH 7.4), 300 mM NaCl, 10 mM imidazole) and elution buffer 150 (50 mM Na-phosphate (pH 7)). 4), 300 mM NaCl, 150 mM imidazole). From this, it is concluded that simple column purification by the His tag of the target product from the culture supernatant is possible.
ｐＩＰＰ−ＰＧ１＃２とｐＰＧ１−ＩＰＰ＃２は、ｐＰＧ１−ＩＰＰ＃１によるＨＥＫ２９３Ｔ細胞の一過性発現と比較し、格段の発現の向上が得られた。また、この結果で、ｐＰＧ１−ＩＰＰ＃２とｐＩＰＰ−ＰＧ１＃２との間でも大きな発現効率の違いが認められているが、この両発現ベクターでは、挿入標的遺伝子配列以外の相違はないので、発現効率には、挿入配列に含まれる要因が関与していると考えられる。 pIPP-PG1 # 2 and pPG1-IPP # 2 showed a marked improvement in expression compared to the transient expression of HEK293T cells by pPG1-IPP # 1. In addition, as a result, a large difference in expression efficiency is recognized between pPG1-IPP # 2 and pIPP-PG1 # 2. However, in both expression vectors, there is no difference other than the insertion target gene sequence. It is considered that factors included in the inserted sequence are involved in the expression efficiency.
＜実施例２および比較例１＞
［検出感度の比較］
１．サンプルの調製
トランスフェリンタンパク質（Ｃａｌｂｉｏｃｈｅｍ、６１６４１９）を滅菌水で溶解（１０ｍｇ／ｍＬ）した。これに等量の２×ＳＤＳ ｓａｍｐｌｅ ｂｕｆｆｅｒ（１２５ｍＭ Ｔｒｉｓ−ＨＣｌ，２０％ ｇｌｙｃｅｒｏｌ，４％ ＳＤＳ，０．０１％ Ｂｒｏｍｏｐｈｅｎｏｌ Ｂｌｕｅ，１０％ ２−ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ，ｐＨ６．８）を加えて熱変性した（１００℃、５分間）。希釈倍率３．１６倍、希釈液１×ＳＤＳ ｓａｍｐｌｅ ｂｕｆｆｅｒを用いて、３．１６ｎｇ／１５μＬ、１ｎｇ／１５μＬ、３１６ｐｇ／１５μＬ、１００ｐｇ／１５μＬ、３１．６ｐｇ／１５μＬ、１０ｐｇ／１５μＬ、３．１６ｐｇ／１５μＬの希釈系列サンプルを作製した。
<Example 2 and Comparative Example 1>
[Detection sensitivity comparison]
1. Sample Preparation Transferrin protein (Calbiochem, 616419) was dissolved in sterile water (10 mg / mL). An equal amount of 2 × SDS sample buffer (125 mM Tris-HCl, 20% glycerol, 4% SDS, 0.01% Bromophenol Blue, 10% 2-mercaptoethanol, pH 6.8) was added to this and heat denatured (100 ° C. 5 minutes). Using a dilution ratio of 3.16 times and a diluent 1 × SDS sample buffer, 3.16 ng / 15 μL, 1 ng / 15 μL, 316 pg / 15 μL, 100 pg / 15 μL, 31.6 pg / 15 μL, 10 pg / 15 μL, 3.16 pg / A 15 μL dilution series sample was made.
２．ウエスタンブロット
ポリアクリルアミドゲルに希釈系列サンプルを１５μＬずつアプライし（３．１６ｎｇ〜３．１６ｐｇ／レーン）、以下の条件でＳＤＳ−ＰＡＧＥ（２００Ｖ定電圧、３０〜３５分間）により分離した。
電気泳動槽：ミニプロティアンＴｅｔｒａセル（Ｂｉｏ−Ｒａｄ）
ポリアクリルアミドゲル：ミニプロティアンＴＧＸゲル１０％（Ｂｉｏ−Ｒａｄ、４５６−１０３５）
泳動バッファ：２５ｍＭ Ｔｒｉｓ，１９２ｍＭ ｇｌｙｃｉｎｅ，０．１％ＳＤＳ，ｐＨ８．３
分子量マーカ：Ｐｒｅｃｉｓｉｏｎ Ｐｌｕｓ Ｐｒｏｔｅｉｎ Ａｌｌ Ｂｌｕｅ Ｓｔａｎｄａｒｄｓ（Ｂｉｏ−Ｒａｄ、１６１−０３７３）
2. Western blot 15 μL of dilution series sample was applied to a polyacrylamide gel (3.16 ng to 3.16 pg / lane), and separated by SDS-PAGE (200 V constant voltage, 30 to 35 minutes) under the following conditions.
Electrophoresis tank: Mini-PROTEAN Tetra cell (Bio-Rad)
Polyacrylamide gel: Mini-PROTEAN TGX gel 10% (Bio-Rad, 456-1035)
Running buffer: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3
Molecular weight marker: Precision Plus Protein All Blue Standards (Bio-Rad, 161-0373)
ブロッティング（２００ｍＡ定電流、４０分間）は、以下の条件で行った。
メンブレン（Ｈｙｂｏｎｄ Ｐメンブレン、ＧＥヘルスケア、ＲＰＮ２０２０Ｆ（１００％メタノールに１分間浸漬した後、蒸留水で５分間洗浄し、転写バッファに浸して平衡化））
転写バッファ（２５ｍＭ Ｔｒｉｓ，１９２ｍＭ ｇｌｙｃｉｎｅ，２０％ Ｍｅｔｈａｎｏｌ，ｐＨ８．３）
Blotting (200 mA constant current, 40 minutes) was performed under the following conditions.
Membrane (Hybond P Membrane, GE Healthcare, RPN2020F (Soaked in 100% methanol for 1 minute, then washed with distilled water for 5 minutes, soaked in transfer buffer and equilibrated))
Transcription buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3)
ブロッティング後のメンブレンをＴＢＳ（２５ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ７．５，１３７ｍＭ ＮａＣｌ，２．７ｍＭ ＫＣｌ）に浸し、室温で３分間振盪し、メンブレンをＴＢＳＴ（２５ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ７．５，１３７ｍＭ ＮａＣｌ，２．７ｍＭ ＫＣｌ，０．１％ Ｔｗｅｅｎ２０）で溶解した５％スキムミルクに浸し、室温で６０分間振盪し、メンブレンをＴＢＳＴに浸し、室温で５分間、４回振盪し、ＴＢＳＴで希釈した１次抗体にメンブレンを浸した（１次抗体：Ｒａｂｂｉｔ ａｎｔｉ−ｈｕｍａｎ Ｔｒａｎｓｆｅｒｒｉｎ ａｎｔｉｂｏｄｙ（ＤＡＫＯ、Ａ００６１）、濃度：４．３μｇ／ｍＬ）。４℃で終夜静置した後、メンブレンをＴＢＳＴに浸し、室温で５分間、４回振盪し、ＴＢＳＴで希釈した下記プロテインＧまたは２次抗体それぞれにメンブレンを浸した。 The membrane after blotting was immersed in TBS (25 mM Tris-HCl, pH 7.5, 137 mM NaCl, 2.7 mM KCl), shaken at room temperature for 3 minutes, and the membrane was TBST (25 mM Tris-HCl pH 7.5, 137 mM NaCl, 2 Soaked in 5% skim milk dissolved in 7 mM KCl, 0.1% Tween20), shaken at room temperature for 60 minutes, soaked in TBST, shaken 4 times at room temperature for 5 minutes, diluted with TBST to the primary antibody. The membrane was immersed (primary antibody: Rabbit anti-human Transfer antibody (DAKO, A0061), concentration: 4.3 μg / mL). After allowing to stand at 4 ° C. overnight, the membrane was immersed in TBST, shaken 4 times at room temperature for 5 minutes, and immersed in each of the following protein G or secondary antibody diluted with TBST.
［実施例２］
ＡＰ標識プロテインＧ（実施例１で調製したＩＰＰ−ＰＧ１＃２） ０．４μg／ｍＬ
［以下、比較例１］
ＡＰ標識プロテインＧ（Ａｂｃａｍ、ａｂ７４６１） ０．４μg／ｍＬ
ＡＰ標識プロテインＧ（Ｒｏｃｋｌａｎｄ、ＰＧ００−０５） ０．４μg／ｍＬ
ＡＰ標識２次抗体（Ｒｏｃｋｌａｎｄ、６１１−１５０２） ０．１μg／ｍＬ
ＡＰ標識２次抗体（ＫＰＬ、４７５１−１５０６） ０．１μg／ｍＬ
ＨＲＰ標識プロテインＧ（Ｒｏｃｋｌａｎｄ、ＰＧ００−０３） ０．１μg／ｍＬ
ＨＲＰ標識プロテインＧ（ｉｎｖｉｔｒｏｇｅｎ、Ｐ−２１０４１） ２．０μg／ｍＬ
ＨＲＰ標識２次抗体（ＧＥヘルスケア、ＮＡ９３４−１ＭＬ） ０．００６μg／ｍＬ
ＨＲＰ標識２次抗体（ＰＩＥＲＣＥ、３１４６２） ０．０２μg／ｍＬ
[Example 2]
AP-labeled protein G (IPP-PG1 # 2 prepared in Example 1) 0.4 μg / mL
[Comparative Example 1]
AP-labeled protein G (Abcam, ab7461) 0.4 μg / mL
AP-labeled protein G (Rockland, PG00-05) 0.4 μg / mL
AP labeled secondary antibody (Rockland, 611502) 0.1 μg / mL
AP labeled secondary antibody (KPL, 4751-1506) 0.1 μg / mL
HRP-labeled protein G (Rockland, PG00-03) 0.1 μg / mL
HRP-labeled protein G (invitrogen, P-21041) 2.0 μg / mL
HRP-labeled secondary antibody (GE Healthcare, NA934-1ML) 0.006 μg / mL
HRP-labeled secondary antibody (PIERCE, 31462) 0.02 μg / mL
室温で６０分間振盪し、メンブレンをＴＢＳＴに浸し、室温で５分間、４回振盪し、メンブレンをハイブリバッグに入れ、発光基質を加えてシーラーでシールした。発光基質は、ＡＰについては、ＣＤＰ−Ｓｔａｒ、ｒｅａｄｙ−ｔｏ−ｕｓｅ（Ｒｏｃｈｅ、２０４１６７７）を用い、ＨＲＰについては、ＥＣＬ Ｐｒｉｍｅ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｅｃｔｉｏｎ Ｒｅａｇｅｎｔ（ＧＥヘルスケア、ＲＰＮ２２３２）を用いた。メンブレンを画像撮影システム（ＣｈｅｍｉＤｏｃ ＸＲＳ ｐｌｕｓシステム、Ｂｉｏ−Ｒａｄ）で撮影した。結果を図４に示す。 The membrane was shaken at room temperature for 60 minutes, soaked in TBST, and shaken 4 times at room temperature for 5 minutes. As the luminescent substrate, CDP-Star and ready-to-use (Roche, 2041677) were used for AP, and ECL Prime Blotting Detection Reagent (GE Healthcare, RPN2232) was used for HRP. The membrane was photographed with an image photographing system (ChemiDoc XRS plus system, Bio-Rad). The results are shown in FIG.
実施例１で調製したＩＰＰ−ＰＧ１＃２は、検出感度が従来の標識プロテインＧより高く、標識２次抗体と同程度であった。 IPP-PG1 # 2 prepared in Example 1 had a detection sensitivity higher than that of the conventional labeled protein G and was similar to that of the labeled secondary antibody.
＜実施例３および比較例２＞
［免疫沈降］
図５および図６に示すように、ｄｉｍｅｒを形成するＤＪ−１タンパク質にタグを付加したＦＬＡＧ−ＤＪ−１とＨＡ−ＤＪ−１を２９３Ｔ細胞に共発現させ、細胞抽出液に抗ＦＬＡＧ抗体（α−ＦＬＡＧ Ｍ２ ａｇａｒｏｓｅ）を加え、ＦＬＡＧ−ＤＪ−１を免疫沈降した。上清を除去した免疫沈降サンプルにＳＤＳ ｓａｍｐｌｅ ｂｕｆｆｅｒ（終濃度：１×）を加えて熱変性（１００℃、５分間）し、同じ量をアプライしたメンブレンを２枚作製してウエスタンブロットを行った。共沈したＨＡ−ＤＪ−１をＨＡ抗体で検出し、一方を実施例１で調製したＩＰＰ−ＰＧ１＃２、他方を蛍光標識２次抗体で検出して比較した。
<Example 3 and Comparative Example 2>
[Immunoprecipitation]
As shown in FIG. 5 and FIG. 6, FLAG-DJ-1 and HA-DJ-1 in which a tag is added to DJ-1 protein forming dimer are co-expressed in 293T cells, and anti-FLAG antibody ( α-FLAG M2 agarose) was added and FLAG-DJ-1 was immunoprecipitated. SDS sample buffer (final concentration: 1 ×) was added to the immunoprecipitated sample from which the supernatant was removed and heat-denatured (100 ° C., 5 minutes), and two membranes to which the same amount was applied were prepared and subjected to Western blotting. . The co-precipitated HA-DJ-1 was detected with the HA antibody, one was IPP-PG1 # 2 prepared in Example 1, and the other was detected with the fluorescently labeled secondary antibody and compared.
上記（実施例２の２．ウエスタンブロット）と同様にして分離、ブロッティングした後、メンブレンをＴＢＳＴで溶解した２．５％スキムミルクに浸し、室温で３０分間振盪し、メンブレンをＴＢＳＴに浸し、室温で５分間、３回振盪し、ＴＢＳＴで希釈した１次抗体にメンブレンを浸した（１次抗体：α−ＨＡ ｒａｂｂｉｔ ｐｏｌｙｃｌｏｎａｌ ａｎｔｉｂｏｄｙ（ＭＢＬ５６１−５、Ｌｏｔ．０５３）、希釈倍率：１，０００倍）。室温で６０分間振盪し、メンブレンをＴＢＳＴに浸し、室温で５分間、３回振盪し、ＴＢＳＴで希釈した以下のプロテインＧまたは２次抗体にメンブレンを浸した。 After separation and blotting in the same manner as described above (2. Western blot in Example 2), the membrane was soaked in 2.5% skim milk dissolved in TBST, shaken at room temperature for 30 minutes, and soaked in TBST. The membrane was shaken 3 times for 5 minutes and immersed in the primary antibody diluted with TBST (primary antibody: α-HA rabbit polyantibody (MBL561-5, Lot. 053), dilution factor: 1,000 times). The membrane was shaken for 60 minutes at room temperature, soaked in TBST, shaken 3 times for 5 minutes at room temperature, and soaked in the following protein G or secondary antibody diluted with TBST.
［実施例３］
ＡＰ標識プロテインＧ（実施例１で調製したＩＰＰ−ＰＧ１＃２） ０．４g／ｍＬ
［比較例２］
Ａｌｅｘａ６８０標識２次抗体（ｉｎｖｉｔｒｏｇｅｎ、Ａ−２１１０９） ０．３μg／ｍＬ
[Example 3]
AP-labeled protein G (IPP-PG1 # 2 prepared in Example 1) 0.4 g / mL
[Comparative Example 2]
Alexa680-labeled secondary antibody (invitrogen, A-21109) 0.3 μg / mL
メンブレンをＴＢＳＴに浸し、室温で５分間、３回振盪し、蒸留水に浸した。ＡＰ標識プロテインＧの方は、メンブレンをハイブリバッグに入れ、発色基質を加えてシーラーでシールし（ＢＣＩＰ／ＮＢＴ溶液、和光純薬工業、０２２−１８２３１）、室温で１０分間静置し、メンブレンを蒸留水に浸し、メンブレンをスキャナで撮影した。Ａｌｅｘａ６８０標識２次抗体の方は、メンブレンをＯｄｙｓｓｅｙ（ＬＩ−ＣＯＲ）で撮影し、蛍光検出した。結果を図７に示す。 The membrane was immersed in TBST, shaken 3 times for 5 minutes at room temperature, and immersed in distilled water. For AP-labeled protein G, place the membrane in a hybrid bag, add a chromogenic substrate, seal with a sealer (BCIP / NBT solution, Wako Pure Chemical Industries, 022-18231), and leave it at room temperature for 10 minutes. Immerse in distilled water and photograph the membrane with a scanner. For the Alexa680-labeled secondary antibody, the membrane was photographed with Odyssey (LI-COR), and fluorescence was detected. The results are shown in FIG.
ＤＪ−１は１８９ａ．ａ．で、ウエスタンブロットする際に軽鎖とほぼ同じ位置に出る。Ａｌｅｘａ６８０標識２次抗体で検出した方には、ＩＰしたサンプル全てで、免疫沈降に用いた抗体の軽鎖を検出してしまい、共沈してきたＨＡ−ＤＪ−１のバンドと重なっているが、ＡＰ標識プロテインＧ（実施例１で調製したＩＰＰ−ＰＧ１＃２）の方では、免疫沈降で使用した抗体の軽鎖は検出されず、目的とするバンドのみの結果となった。また、単なるｒａｂｂｉｔ ＩｇＧも熱変性してから泳動した。Ａｌｅｘａ６８０標識２次抗体の方では重鎖が検出されているが、ＡＰ標識プロテインＧ（実施例１で調製したＩＰＰ−ＰＧ１＃２）の方では検出されなかった。 DJ-1 is 189a. a. Thus, when Western blotting is performed, it comes out at almost the same position as the light chain. For those detected with Alexa680-labeled secondary antibody, the IP light chain detected in all IP samples was overlapped with the co-precipitated HA-DJ-1 band, In the case of AP-labeled protein G (IPP-PG1 # 2 prepared in Example 1), the light chain of the antibody used in the immunoprecipitation was not detected, and only the target band was obtained. In addition, mere rabbit IgG was also electrophoresed after heat denaturation. A heavy chain was detected in the Alexa680-labeled secondary antibody, but not in AP-labeled protein G (IPP-PG1 # 2 prepared in Example 1).
このように、実施例１で調製したＩＰＰ−ＰＧ１＃２を用いると、免疫沈降のキャプチャー抗体が検出されなかった。 Thus, when IPP-PG1 # 2 prepared in Example 1 was used, no immunoprecipitation capture antibody was detected.
＜実施例４および比較例３＞
［内在性ＩｇＧの検出の有無の確認］
以下の方法により、内在性ＩｇＧの検出の有無を比較した。結果を図８に示す。
<Example 4 and Comparative Example 3>
[Confirmation of detection of endogenous IgG]
The presence or absence of endogenous IgG was compared by the following method. The results are shown in FIG.
１．サンプルの調製
ラット脳組織抽出液（２ｍｇ／ｍＬ）に等量の２×ＳＤＳ Ｓａｍｐｌｅ ｂｕｆｆｅｒを加えて熱変性した。
1. Sample Preparation An equal amount of 2 × SDS Sample buffer was added to rat brain tissue extract (2 mg / mL) and heat denatured.
２．ウエスタンブロット
ポリアクリルアミドゲルにサンプルを２μＬ（２μｇ）、１０μＬ（１０μｇ）アプライし、実施例２の「２．ウエスタンブロット」と同様の条件でウエスタンブロットを行った。
2. Western blot 2 μL (2 μg) and 10 μL (10 μg) of the sample were applied to a polyacrylamide gel, and Western blotting was performed under the same conditions as “2. Western blot” in Example 2.
［実施例４］
ＡＰ標識プロテインＧ（実施例１で調製したＩＰＰ−ＰＧ１＃２）
［以下、比較例３］
ＡＰ標識プロテインＧ（Ｒｏｃｋｌａｎｄ、ＰＧ００−０５）
ＡＰ標識２次抗体（ＫＰＬ、４７５１−１５０６）
[Example 4]
AP-labeled protein G (IPP-PG1 # 2 prepared in Example 1)
[Comparative Example 3]
AP-labeled protein G (Rockland, PG00-05)
AP labeled secondary antibody (KPL, 4751-1506)
実施例１で調製したＩＰＰ−ＰＧ１＃２では、内在性ＩｇＧが検出されなかった。 In IPP-PG1 # 2 prepared in Example 1, endogenous IgG was not detected.
＜実施例５＞
［免疫染色］
マウス組織（腎臓、脾臓、肝臓、心臓）をアレイにしたブロックを作製し、下記ＡＰ標識プロテインＧまたはＡＰ標識２次抗体を用いて表３に示す手順により、発色基質としてＮＢＴ／ＢＣＩＰにより免疫染色を行った。マウス心臓についての免疫染色の結果を図９に示す。
<Example 5>
[Immunostaining]
A block in which mouse tissues (kidney, spleen, liver, heart) were arrayed was prepared and immunostained with NBT / BCIP as a chromogenic substrate by the procedure shown in Table 3 using the following AP-labeled protein G or AP-labeled secondary antibody. Went. The results of immunostaining for the mouse heart are shown in FIG.
（１）１次抗体：なし、ＡＰ標識プロテインＧ：実施例１で調製したＩＰＰ−ＰＧ１＃２ ２ｕｇ／ｍＬ
（２）１次抗体：Ａｃｔｉｎ（Ａｎｔｉ−Ａｃｔｉｎ ａｎｔｉｂｏｄｙ ｐｒｏｄｕｃｅｄ ｉｎ ｒａｂｂｉｔ、Ａ２０６６、Ｓｉｇｍａ）（１／２００）、ＡＰ標識プロテインＧ：実施例１で調製したＩＰＰ−ＰＧ１＃２ ２ｕｇ／ｍＬ
（３）１次抗体：Ａｃｔｉｎ（１／２００）、標識２次抗体：ＫＰＬ、４７５１−１５０６ ２ｕｇ／ｍＬ
(1) Primary antibody: None, AP-labeled protein G: IPP-PG1 # 2 2 ug / mL prepared in Example 1
(2) Primary antibody: Actin (Anti-actin antibody produced in rabbit, A2066, Sigma) (1/200), AP-labeled protein G: IPP-PG1 # 2 prepared in Example 1 2 ug / mL
(3) Primary antibody: Actin (1/200), labeled secondary antibody: KPL, 4751-1506 2 ug / mL
このように、実施例１で調製したＩＰＰ−ＰＧ１＃２を用いると、ＡＰ標識２次抗体と同様のシグナルが得られ、非特異的結合によるバックグラウンドの上昇はほとんど見られなかった。 Thus, when IPP-PG1 # 2 prepared in Example 1 was used, a signal similar to that of the AP-labeled secondary antibody was obtained, and the background was hardly increased due to non-specific binding.
以上の通り、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインのうち少なくとも１つを含むタンパク質ドメインと、ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼとを融合させることにより、汎用性が高く、検出感度が高く、かつ安定性の高いタンパク質検出用融合タンパク質が得られた。また、そのタンパク質検出用融合タンパク質を用いて、汎用性が高く、検出感度が高いタンパク質の検出方法を提供することができた。 As described above, a protein domain containing at least one of the C2 domain of protein G and the C3 domain of protein G, and a fused alkaline phosphatase in which partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase are fused. By fusing, a fusion protein for protein detection having high versatility, high detection sensitivity, and high stability was obtained. Moreover, using the fusion protein for protein detection, a method for detecting a protein having high versatility and high detection sensitivity could be provided.
Claims (4)
前記タンパク質ドメインが、プロテインＡのＢドメイン、プロテインＧのＣ２ドメインおよびプロテインＧのＣ３ドメインが結合されたものであることを特徴とするタンパク質検出用融合タンパク質。 A fusion protein for protein detection according to claim 1,
A protein detection fusion protein, wherein the protein domain is a combination of a protein A B domain, a protein G C2 domain, and a protein G C3 domain.
前記融合型アルカリホスファターゼが、
ヒト胎盤型アルカリホスファターゼとヒト小腸型アルカリホスファターゼそれぞれの部分配列を融合した融合型アルカリホスファターゼＩＰＰまたはＩＩＰであることを特徴とするタンパク質検出用融合タンパク質。 A fusion protein for protein detection according to claim 1,
The fused alkaline phosphatase is
A fusion protein for protein detection, which is a fusion alkaline phosphatase IPP or IIP in which partial sequences of human placental alkaline phosphatase and human small intestine alkaline phosphatase are fused.
請求項１〜３のいずれか１項に記載のタンパク質検出用融合タンパク質と、対象物中に存在するタンパク質とを直接または間接的に結合させ、結合している前記タンパク質検出用融合タンパク質のアルカリホスファターゼ部分を標識部分として検出することを特徴とするタンパク質の検出方法。 A protein detection method comprising:
The protein detection fusion protein according to any one of claims 1 to 3 and the protein present in the object are directly or indirectly bound to each other, and the protein detection fusion protein alkaline phosphatase is bound thereto. A method for detecting a protein, wherein the portion is detected as a labeling portion.
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| WO2015056659A1 (en) * | 2013-10-18 | 2015-04-23 | 国立大学法人九州大学 | Fusion protein for protein detection, and method for detecting protein |
| WO2016017037A1 (en) * | 2014-08-01 | 2016-02-04 | 株式会社ビークル | Virus-like particle to be used in immunoassay method, blocking agent to be used therein and kit comprising same |
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| WO2015056659A1 (en) * | 2013-10-18 | 2015-04-23 | 国立大学法人九州大学 | Fusion protein for protein detection, and method for detecting protein |
| US9976129B2 (en) | 2013-10-18 | 2018-05-22 | Kyushu University, National University Corporation | Fusion protein for protein detection, and method for detecting protein |
| WO2016017037A1 (en) * | 2014-08-01 | 2016-02-04 | 株式会社ビークル | Virus-like particle to be used in immunoassay method, blocking agent to be used therein and kit comprising same |
| JP5867890B1 (en) * | 2014-08-01 | 2016-02-24 | 株式会社ビークル | Virus-like particles used in immunoassay, blocking agents used therefor, and kits containing these |
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