JP2021100965A - RNase inhibitor composition - Google Patents
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本発明は、ＲＮアーゼ（以下「ＲＮａｓｅ」という。）阻害剤の凝集を抑制するための組成物に関する。特に、界面活性剤を用いることを特徴とするＲＮａｓｅ阻害剤の凝集を抑制することができる組成物に関する。 The present invention relates to a composition for suppressing aggregation of an RNase (hereinafter referred to as "RNase") inhibitor. In particular, the present invention relates to a composition capable of suppressing aggregation of an RNase inhibitor, which is characterized by using a surfactant.
ＲＮａｓｅ阻害剤はＲＮＡ分解酵素であるＲＮａｓｅの阻害剤として、遺伝子検査試薬や研究用試薬（例えば、逆転写ポリメラーゼ連鎖反応、ＲＴ−ＰＣＲなど）等に含まれる成分として重要であり、広く使用されている（特許文献２）。しかしながら、ＲＮａｓｅ阻害剤は酸化されやすく、ジスルフィド結合を形成しやすい特徴を持つタンパク質である。そのため、ＲＮａｓｅ阻害剤を含む溶液は、酸化によりジスルフィド結合が形成されやすく、分子内部ジスルフィド結合または外部ジスルフィド結合により凝集が発生し、アクティブサイトがブロックされ活性が低下する可能性がある。あるいは水溶液中の疎水性相互作用や表面張力により凝集が形成される可能性がある。さらには、濃縮や混合等による物理的動作や凍結融解等による濃度勾配の発生により凝集が発生する可能性がある。また、凝集の発生は、ＲＮａｓｅ阻害剤を含む水溶液中のＲＮａｓｅ阻害剤の濃度が高濃度であるほど発生しやすい。試薬の調製や使用する際に凝集物が生じることは、作業上あるいは測定上好ましくなく、また、凝集によりアクティブサイトがブロックされ活性が減少することは、試薬の性能に影響を及ぼすため許容されない（非特許文献１）。 The RNase inhibitor is important as an inhibitor of RNase, which is an RNA-degrading enzyme, and is important as a component contained in genetic test reagents and research reagents (for example, reverse transcription polymerase chain reaction, RT-PCR, etc.), and is widely used. (Patent Document 2). However, RNase inhibitors are proteins that are prone to oxidation and form disulfide bonds. Therefore, in a solution containing an RNase inhibitor, disulfide bonds are likely to be formed by oxidation, and aggregation may occur due to internal disulfide bonds or external disulfide bonds, blocking active sites and reducing activity. Alternatively, agglutination may be formed due to hydrophobic interaction or surface tension in the aqueous solution. Furthermore, agglomeration may occur due to physical operation such as concentration or mixing, or generation of a concentration gradient due to freezing and thawing. Further, the occurrence of aggregation is more likely to occur as the concentration of the RNase inhibitor in the aqueous solution containing the RNase inhibitor is higher. The formation of agglomerates during the preparation and use of the reagent is not desirable for work or measurement, and it is unacceptable for the agglomeration to block active sites and reduce activity, as this affects the performance of the reagent ( Non-Patent Document 1).
従って、ＲＮａｓｅ阻害剤を含む水溶液においては、凝集を抑制することが重要であるが、従来この凝集物の生成を効果的に防止することのできる組成は知られていなかった。 Therefore, in an aqueous solution containing an RNase inhibitor, it is important to suppress aggregation, but conventionally, a composition capable of effectively preventing the formation of this aggregate has not been known.
これまでにタンパク質の凝集を抑制する方法の例として、抗体を含む溶液に界面活性剤を添加することが提案されている（特許文献１）。この方法では限外濾過において界面活性剤を添加することで凝集物の生成や白濁化を抑制し、抗体を含む診断薬や治療剤、特に注射剤に効果を発揮している。このように、水溶液中のタンパク質凝集を抑制することは重要であるが、凝集の発生しやすい水溶液中のＲＮａｓｅの阻害剤が高い場合において、凝集を抑制する方法は知られていなかった。また上述のＲＴ−ＰＣＲのような遺伝子検査等において用いられるＲＮａｓｅ阻害剤の場合においては、反応阻害を引き起こすことなく、ＲＮａｓｅ阻害剤の凝集を効果的に抑制する方法は知られていなかった。 So far, as an example of a method for suppressing protein aggregation, it has been proposed to add a surfactant to a solution containing an antibody (Patent Document 1). In this method, the formation of aggregates and clouding are suppressed by adding a surfactant in the ultrafiltration, and it is effective for diagnostic agents and therapeutic agents containing antibodies, especially injections. As described above, it is important to suppress protein aggregation in an aqueous solution, but a method for suppressing aggregation has not been known when the inhibitor of RNase in an aqueous solution in which aggregation is likely to occur is high. Further, in the case of an RNase inhibitor used in genetic testing such as RT-PCR described above, a method for effectively suppressing the aggregation of the RNase inhibitor without causing reaction inhibition has not been known.
本発明は、水溶液中におけるＲＮａｓｅ 阻害剤の凝集を効果的に防止可能であり、かつ、ＲＴ−ＰＣＲのような遺伝子検査等において反応阻害を引き起こすことのない組成物の提供を目的とする。 An object of the present invention is to provide a composition capable of effectively preventing aggregation of an RNase inhibitor in an aqueous solution and not causing reaction inhibition in a genetic test such as RT-PCR.
本発明者らは、上記事情に鑑み鋭意研究を行った結果、ＲＮａｓｅ阻害剤を含む水溶液において、界面活性剤を添加することにより、ＲＴ−ＰＣＲのような遺伝子解析において悪影響を及ぼすことなく、ＲＮａｓｅ阻害剤の凝集を抑制できることを見出し、本発明を完成するに至った。 As a result of diligent research in view of the above circumstances, the present inventors have conducted RNase in an aqueous solution containing an RNase inhibitor without adversely affecting gene analysis such as RT-PCR by adding a surfactant. We have found that the aggregation of inhibitors can be suppressed, and have completed the present invention.
本発明は、以下のような構成からなる。
項１．ＲＮａｓｅ（ＲＮａｓｅ）阻害剤を含む水溶液中において界面活性剤を含有せしめることを特徴とするＲＮａｓｅ阻害剤組成物。
項２．水溶液中における前期ＲＮａｓｅ阻害剤の濃度が５〜２００Ｕ／μｌである項１に記載のＲＮａｓｅ阻害剤組成物。
項３．水溶液中における前記界面活性剤の濃度が０．００１〜１％である項１または２記載のＲＮａｓｅ阻害剤組成物。
項４．水溶液中における前記界面活性剤の濃度が０．０１〜０．１％である項３に記載のＲＮａｓｅ阻害剤組成物。
項５．水溶液中に含まれる前記界面活性剤が非イオン性界面活性剤である項１から４のいずれかに記載のＲＮａｓｅ阻害剤組成物。
項６．水溶液中における前記界面活性剤がＳＤＳ、ＣＨＡＰＳ、コール酸ナトリウムおよびデオキシコール酸ナトリウムからなる群よりから選ばれる少なくとも１種類の界面活性剤である項１から４のいずれかに記載のＲＮａｓｅ阻害剤組成物。
項７．水溶液中における前記界面活性剤がＴｗｅｅｎ−２０、ノニデット Ｐ−４０、ＴｒｉｔｏｎＸ−１００、Ｂｒｉｇ３５、ＳＤＳ、ＣＨＡＰＳ、コール酸ナトリウムおよびデオキシコール酸ナトリウムからなる群より選ばれる少なくとも１種類の界面活性剤である項１から４のいずれかに記載のＲＮａｓｅ阻害剤組成物。
項８．水溶液中のＲＮａｓｅ阻害剤凝集を抑制するために用いることを特徴とする項１から７のいずれかに記載のＲＮａｓｅ阻害剤組成物。
項９．項１から８のいずれかに記載のＲＮａｓｅ阻害剤組成物を含んでなることを特徴とするＲＴ−ＰＣＲ反応用キット。
The present invention has the following configuration.
Item 1. An RNase inhibitor composition comprising containing a surfactant in an aqueous solution containing an RNase (RNase) inhibitor.
Item 2. Item 2. The RNase inhibitor composition according to Item 1, wherein the concentration of the early RNase inhibitor in the aqueous solution is 5 to 200 U / μl.
Item 3. Item 2. The RNase inhibitor composition according to Item 1 or 2, wherein the concentration of the surfactant in the aqueous solution is 0.001 to 1%.
Item 4. Item 3. The RNase inhibitor composition according to Item 3, wherein the concentration of the surfactant in the aqueous solution is 0.01 to 0.1%.
Item 5. Item 2. The RNase inhibitor composition according to any one of Items 1 to 4, wherein the surfactant contained in the aqueous solution is a nonionic surfactant.
Item 6. Item 2. The RNase inhibitor composition according to any one of Items 1 to 4, wherein the surfactant in an aqueous solution is at least one surfactant selected from the group consisting of SDS, CHAPS, sodium cholic acid and sodium deoxycholate. Stuff.
Item 7. The surfactant in an aqueous solution is at least one surfactant selected from the group consisting of Tween-20, Nonidet P-40, Triton X-100, Brig35, SDS, CHAPS, sodium cholic acid and sodium deoxycholate. Item 4. The RNase inhibitor composition according to any one of Items 1 to 4.
Item 8. Item 2. The RNase inhibitor composition according to any one of Items 1 to 7, which is used for suppressing RNase inhibitor aggregation in an aqueous solution.
Item 9. A kit for RT-PCR reaction, which comprises the RNase inhibitor composition according to any one of Items 1 to 8.
本発明により、ＲＮａｓｅ阻害剤の凝集による作業上の問題や活性の低下を防ぐことができる。ＲＮａｓｅ阻害剤を含む水溶液を、ＲＮａｓｅ阻害剤を含む水溶液を保管する場合においても凝集を抑制することが可能である。高濃度のＲＮａｓｅ阻害剤を含む水溶液では凝集が発生しやすいため、よりその効果を発揮する。また、本発明における及び組成は、遺伝子検査薬や研究用試薬の性能に対して影響を与えることがない点で、非常に有用である。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to prevent work problems and decrease in activity due to aggregation of RNase inhibitors. Aggregation can be suppressed even when the aqueous solution containing an RNase inhibitor is stored in the aqueous solution containing an RNase inhibitor. An aqueous solution containing a high concentration of an RNase inhibitor tends to cause aggregation, so that the effect is more exerted. In addition, the and composition in the present invention are very useful in that they do not affect the performance of genetic test agents and research reagents.
本発明は、ＲＮａｓｅ阻害剤を含有する水溶液に界面活性剤を添加することを特徴とするものであり、ＲＮａｓｅ阻害剤の凝集が抑制されたＲＮａｓｅ阻害剤組成物を提供する。 The present invention is characterized by adding a surfactant to an aqueous solution containing an RNase inhibitor, and provides an RNase inhibitor composition in which aggregation of the RNase inhibitor is suppressed.
本発明における凝集とは、ＲＮａｓｅ阻害剤を含む水溶液中に目視にて判別される不溶物のことである。凝集の発生要因は特に限定されるものではない。水溶液中に目視される凝集はＲＮａｓｅ阻害剤を保管することで発生するものを含み、混合等による物理的動作によるものも含まれる。具体的には、ＲＮａｓｅ阻害剤を含む水溶液をＭＩＸ−ＲＯＴＡＲ ＭＲ−５（ＡＳＯＮＥ）を用いて、回転数４０ｒｐｍで６時間撹拌させたときに、水溶液中に目視にて物質が判別される場合における該物質のことである。 Aggregation in the present invention is an insoluble matter that is visually discriminated in an aqueous solution containing an RNase inhibitor. The factors that cause agglomeration are not particularly limited. Aggregation visually observed in the aqueous solution includes those generated by storing the RNase inhibitor, and also includes those caused by physical operation such as mixing. Specifically, when an aqueous solution containing an RNase inhibitor is stirred using MIX-ROTAR MR-5 (AS ONE) at a rotation speed of 40 rpm for 6 hours, a substance is visually identified in the aqueous solution. It is the substance.
本発明におけるＲＮａｓｅ阻害剤としては、特に限定されないが、例えばヒト胎盤由来、ラット肺由来やブタ肝臓由来のタンパク質が挙げられる。また、大腸菌等の宿主を用いて発現させた組換え体から取得することが可能である。その方法としては、ＲＮａｓｅ阻害剤遺伝子の単離、プラスミドの構築、宿主への導入および細胞融解と遠心によるＲＮａｓｅ阻害剤タンパク質単離のステップを含む方法がある。この方法には単離したＲＮａｓｅ阻害剤タンパク質化学試薬による可溶化、精製のステップが含まれる（特許文献２参照）。 The RNase inhibitor in the present invention is not particularly limited, and examples thereof include proteins derived from human placenta, rat lung, and porcine liver. It can also be obtained from a recombinant expressed using a host such as Escherichia coli. Methods include isolation of the RNase inhibitor gene, plasmid construction, introduction into the host, and isolation of the RNase inhibitor protein by cell thawing and centrifugation. This method includes solubilization and purification steps with an isolated RNase inhibitor protein chemical reagent (see Patent Document 2).
本発明における界面活性剤とは、特に限定されるものではない。界面活性剤の種類としては、イオン性界面活性剤、非イオン性界面活性剤のいずれであっても良い。具体的には、特に限定されないが、Ｔｗｅｅｎ−２０、ノニデット Ｐ−４０、ＴｒｉｔｏｎＸ−１００、Ｂｒｉｇ３５、ＳＤＳ、ＣＨＡＰＳ、コール酸ナトリウム、デオキシコール酸ナトリウム、Ｄｅｏｘｙ−ＢＩＧＣＨＡＰ 、ＢＩＧＣＨＡＰ、ＮＩＫＫＯＬ ＢＬ−９ＥＸ、ＭＥＧＡ−８、ＭＥＧＡ−９、ＭＥＧＡ−１０、Ｔｒｉｔｏｎ Ｘ−１１４、Ｔｗｅｅｎ−４０、Ｂｒｉｇ５８、ＮＰ−４０、ＤＤＭ、Ｔｗｅｅｎ−８０、サルコシル、尿素、Ｐｏｌｙｏｘｙｅｔｈｙｌｅｎｅ（２０）Ｓｏｒｂｉｔａｎ Ｔｒｉｏｌｅａｔｅ、ＩＧＥＰＡＬ ＣＡ−６３０、Ｄｉｇｉｔｏｎｉｎ、サポニン、ｎ−Ｈｅｐｔｙｌ−β−Ｄ−ｔｈｉｏｇｌｕｃｏｐｙｒａｎｏｓｉｄｅ、ｎ−Ｄｏｄｅｃｙｌ−β−Ｄ−ｍａｌｔｏｐｙｒａｎｏｓｉｄｅ、ｎ−Ｏｃｔｙｌ−β−Ｄ−ｔｈｉｏｇｌｕｃｏｐｙｒａｎｏｓｉｄｅ、ｎ−Ｎｏｎｙｌ−β−Ｄ−ｔｈｉｏｍａｌｔｏｓｉｄｅ、Ｏｃｔｙｌ−β−Ｄ−ｇｌｕｃｏｓｉｄｅ、Ｚｗｉｔｔｅｒｇｅｎｔ ３−１２、Ｄｏｄｅｃｙｌ−β−Ｄ−ｍａｌｔｏｓｉｄｅ等が好ましい。より好ましい界面活性剤としては、Ｔｗｅｅｎ−２０、ノニデット Ｐ−４０、ＴｒｉｔｏｎＸ−１００、Ｂｒｉｇ３５、ＳＤＳ、ＣＨＡＰＳ、コール酸ナトリウム、及びデオキシコール酸ナトリウム等が例示される。これらの界面活性剤は、単独で用いても良いし、複数種を組み合わせて用いても良い。 The surfactant in the present invention is not particularly limited. The type of surfactant may be either an ionic surfactant or a nonionic surfactant. Specifically, but not particularly limited, Tween-20, Nonidet P-40, TritonX-100, Brig35, SDS, CHAPS, sodium cholic acid, sodium deoxycholate, Deoxy-BIGCHAP, BIGCHAP, NIKKOL BL-9EX, MEGA. -8, MEGA-9, MEGA-10, Triton X-114, Tween-40, Brig58, NP-40, DDM, Tween-80, sarcosyl, urea, Polysorbate (20) Polysorbate, IGEPAL CA-630, Digitonin, Saponin, n-Heptyl-β-D-thioglucopylanoside, n-Dodecyl-β-D-maltopylanoside, n-Octyl-β-D-thioglucopylanoside, n-Nonyl-β-d-dyl Zwittergent 3-12, Dodecyl-β-D-maltoside and the like are preferable. More preferred surfactants include Tween-20, Nonidet P-40, Triton X-100, Brig35, SDS, CHAPS, sodium cholic acid, sodium deoxycholate and the like. These surfactants may be used alone or in combination of two or more.
本発明におけるＲＮａｓｅ阻害剤を含む水溶液中における界面活性剤の濃度は、凝集抑制効果やＲＴ−ＰＣＲ等の反応に用いられる際の影響などの観点を考慮すると、０．００１から１％であることが好ましい。より好ましくは０．００５から０，０５％であり、更に好ましくは０．０１％から０．１％である。 The concentration of the surfactant in the aqueous solution containing the RNase inhibitor in the present invention is 0.001 to 1% in consideration of the aggregation inhibitory effect and the influence when used in a reaction such as RT-PCR. Is preferable. It is more preferably 0.005 to 0.05%, still more preferably 0.01% to 0.1%.
ＲＮａｓｅ阻害剤は、ｃＤＮＡ合成やｉｎ ｖｉｔｒｏ Ｔｒａｎｓｌａｔｉｏｎ等、ＲＮＡを使用するあらゆる実験におけるＲＮＡの安定化、ＲＮＡプローブの作製やｉｎ ｖｉｔｒｏ Ｔｒａｎｓｃｒｉｐｔｉｏｎ等、ＲＮＡを産生する種々の実験におけるＲＮＡの安定化において広く使用される。 RNase inhibitors are widely used in RNA stabilization in all experiments that use RNA, such as cDNA synthesis and in vitro translation, in RNA stabilization in various RNA-producing experiments, such as RNA probe fabrication and in vitro translation. Will be done.
本発明におけるＲＮａｓｅ阻害剤を含む水溶液中のＲＮａｓｅ阻害剤の濃度としては、好ましくは５〜２００Ｕ／μｌ、より好ましくは１５〜６０Ｕ／μｌ、さらに好ましくは２０〜４０Ｕ／μｌであるが、特に限定されない。ここで、１Ｕとは、５ｎｇのＲＮａｓｅの活性を５０％阻害するのに必要なＲＮａｓｅ阻害剤量である。酵素活性の測定方法としては、レート法を用いる。レート法とは目的成分と試薬を反応させて、その反応が進行しているときの速度を単位時間当たりの吸光度変化量として測定し、目的成分を定量する方法である。本発明における測定は、反応温度２５℃で、ｃｙｃｌｉｃ ２，３’−ＣＭＰ の加水分解をＲＮａｓｅの一つであるＲＮａｓｅを阻害する効率を２８６ｎｍの吸光度変化で測定を行う。 The concentration of the RNase inhibitor in the aqueous solution containing the RNase inhibitor in the present invention is preferably 5 to 200 U / μl, more preferably 15 to 60 U / μl, still more preferably 20 to 40 U / μl, but is particularly limited. Not done. Here, 1U is the amount of RNase inhibitor required to inhibit the activity of 5 ng of RNase by 50%. The rate method is used as a method for measuring the enzyme activity. The rate method is a method in which a target component is reacted with a reagent, the rate at which the reaction is proceeding is measured as the amount of change in absorbance per unit time, and the target component is quantified. In the measurement in the present invention, the efficiency of inhibiting RNase, which is one of RNase, for hydrolysis of cyclic 2,3'-CMP at a reaction temperature of 25 ° C. is measured by a change in absorbance at 286 nm.
また、ＲＮａｓｅ阻害剤の形状バッファーとしては、例えばＨｅｐｅｓ−ＫＯＨ、Ｈｅｐｅｓ−ＮａＯＨ、Ｔｒｉｓ−ＨＣｌなどが挙げられる。バッファー終濃度は特に限定されないが、１０ｍＭから１００ｍＭ程度が好ましく、２０ｍＭから５０ｍＭ程度がより好ましい。バッファーのｐＨは４から９程度が好ましく、５から８程度がより好ましい。更には終濃度、１ｍＭから２００ｍＭのＫＣｌ、１ｍＭから１００ｍＭのＤＴＴ、３０から７０％のＧｌｙｃｅｒｏｌ等を含んでも良い。 Examples of the shape buffer of the RNase inhibitor include Hepes-KOH, Hepes-NaOH, Tris-HCl and the like. The final buffer concentration is not particularly limited, but is preferably about 10 mM to 100 mM, more preferably about 20 mM to 50 mM. The pH of the buffer is preferably about 4 to 9, more preferably about 5 to 8. Further, a final concentration of 1 mM to 200 mM KCl, 1 mM to 100 mM DTT, 30 to 70% Glycerol and the like may be contained.
本発明の別な態様としては、上述したようなＲＮａｓｅ阻害剤を含む逆転写反応用組成物である。更には、上述したようなＲＮａｓｅ阻害剤を含むＲＴ−ＰＣＲ反応用組成物が挙げられる。これらの組成物を含むＲＴ−ＰＣＲ反応用試薬またはＲＴ−ＰＣＲ反応用キットであってもよい。逆転写反応に必要なものとして、逆転写酵素を存在させる。ＲＴ−ＰＣＲ反応用組成物を構成するものとしては、逆転写酵素、ＤＮＡポリメラーゼ、プライマーとなるオリゴヌクレオチド、ジデオキシヌクレオシド三リン酸（ｄＮＴＰｓ）、抗ＤＮＡポリメラーゼ抗体、反応緩衝剤、マグネシウムイオン等の金属イオンなどを、実施する核酸増幅方法により、必要に応じて存在させる。 Another aspect of the present invention is a composition for reverse transcription reaction containing an RNase inhibitor as described above. Further, a composition for RT-PCR reaction containing an RNase inhibitor as described above can be mentioned. It may be an RT-PCR reaction reagent or an RT-PCR reaction kit containing these compositions. Reverse transcriptase is present as required for the reverse transcription reaction. The composition for RT-PCR reaction includes reverse transcriptase, DNA polymerase, oligonucleotide as a primer, dideoxynucleoside triphosphates (dNTPs), anti-DNA polymerase antibody, reaction buffer, and metals such as magnesium ion. Ions and the like are present as needed by the nucleic acid amplification method to be carried out.
リアルタイムＲＴ−ＰＣＲに用いる場合は、核酸増幅を経時的に確認するため、さらにＳＹＢＲ ＧｒｅｅｎＩ（登録商標）やＥｖａ Ｇｒｅｅｎ（登録商標）などの蛍光色素や蛍光標識をしたプローブを必要に応じて存在させる。また、核酸増幅を経時的に確認するため、さらにＳＹＢＲ ＧｒｅｅｎＩ（登録商標）やＥｖａ Ｇｒｅｅｎ（登録商標）などの蛍光色素や蛍光標識をしたプローブを必要に応じて存在させる。 When used for real-time RT-PCR, fluorescent dyes such as SYBR Green I® and Eva Green® and fluorescently labeled probes should be present as needed to confirm nucleic acid amplification over time. .. Further, in order to confirm nucleic acid amplification over time, a fluorescent dye such as SYBR Green I (registered trademark) or Eva Green (registered trademark) or a fluorescently labeled probe is present as needed.
以下、実施例に基づき本発明をより具体的に説明する。もっとも、本発明は、下記実施例により、特に限定されるものではない。 Hereinafter, the present invention will be described in more detail based on Examples. However, the present invention is not particularly limited by the following examples.
（実施例１）
界面活性剤の添加検討
（１）ＲＮａｓｅ阻害剤を含む水溶液の作製
特許文献２に基づき大腸菌を宿主としてＲＮａｓｅ阻害剤を発現させ、精製することによりＲＮａｓｅ阻害剤を取得した。取得したＲＮａｓｅ阻害剤の活性を、レート法を用いて測定し、１、５、２０、４０、２００Ｕ／μｌのＲＮａｓｅ阻害剤を含む、以下の組成からなる水溶液を作製した。
２０ｍＭ Ｈｅｐｅｓ−ＫＯＨ（ｐＨ７．６）
５０ｍＭ ＫＣｌ
８ｍＭ ＤＴＴ
５０％ Ｇｌｙｃｅｒｏｌ
(Example 1)
Examination of Addition of Surfactant (1) Preparation of Aqueous Solution Containing RNase Inhibitor Based on Patent Document 2, an RNase inhibitor was obtained by expressing and purifying an RNase inhibitor using Escherichia coli as a host. The activity of the obtained RNase inhibitor was measured using the rate method to prepare an aqueous solution having the following composition containing 1, 5, 20, 40, and 200 U / μl of the RNase inhibitor.
20 mM Hepes-KOH (pH 7.6)
50 mM KCl
8 mM DTT
50% Glycerol
（２）界面活性剤の添加
作製した１、５、２０、４０、２００Ｕ／μｌのＲＮａｓｅ阻害剤を含む水溶液１ｍＬに対して各界面活性剤の終濃度が１〜０．００００１％になるように表１に示すように調整した。その後、調製したＲＮａｓｅ阻害剤を含む水溶液を１５ｍＬチューブに入れ、ＭＩＸ−ＲＯＴＡＲ ＭＲ−５（ＡＳＯＮＥ）にて回転数４０ｒｐｍにて６時間撹拌し、目視により凝集の有無を判別した。結果を表２および表３に示す。凝集が見られない場合を○、凝集が見られる場合を×を表記する。
(2) Addition of Surfactant The final concentration of each surfactant is 1 to 0.00001% with respect to 1 mL of the prepared aqueous solution containing 1, 5, 20, 40, and 200 U / μl of RNase inhibitor. The adjustment was made as shown in Table 1. Then, the prepared aqueous solution containing an RNase inhibitor was placed in a 15 mL tube, stirred with MIX-ROTAR MR-5 (AS ONE) at a rotation speed of 40 rpm for 6 hours, and the presence or absence of aggregation was visually determined. The results are shown in Tables 2 and 3. If no agglomeration is observed, mark ○, and if agglomeration is observed, mark ×.
（３）結果
４０Ｕ／μｌのＲＮａｓｅ阻害剤を含む水溶液に界面活性剤を添加した結果を表２、表３に示す。界面活性剤を添加しない場合は凝集が確認されたが、ＲＮａｓｅ阻害剤を含む水溶液に対して界面活性剤を０．００１〜１％添加することで凝集が発生しないことが確認できた。同様の結果が、５、２０、２００Ｕ／μｌのＲＮａｓｅ阻害剤を含む水溶液でも得られた。
(3) Results Tables 2 and 3 show the results of adding a surfactant to an aqueous solution containing a 40 U / μl RNase inhibitor. Aggregation was confirmed when no surfactant was added, but it was confirmed that aggregation did not occur when 0.001 to 1% of the surfactant was added to the aqueous solution containing the RNase inhibitor. Similar results were obtained with aqueous solutions containing 5, 20, 200 U / μl of RNase inhibitors.
表４は凝集有無と水溶液中のＲＮａｓｅ阻害剤濃度と界面活性剤（Ｔｗｅｅｎ−２０）濃度の関係性を示す。１Ｕ／μｌのＲＮａｓｅ阻害剤を含む水溶液では界面活性剤を添加しない場合でも凝集が確認されない。水溶液中の界面活性剤の濃度が５Ｕ／μｌ以上であると、界面活性剤を含まない場合や濃度が低い場合に凝集が発生した。これらの結果から、水溶液中のＲＮａｓｅ阻害剤の濃度が高いほど凝集が発生しやすいことが示唆され、界面活性剤を添加することで凝集の発生の抑制可能であるといえる。 Table 4 shows the relationship between the presence or absence of aggregation, the concentration of the RNase inhibitor in the aqueous solution, and the concentration of the surfactant (Tween-20). In an aqueous solution containing 1 U / μl of an RNase inhibitor, no aggregation is confirmed even without the addition of a surfactant. When the concentration of the surfactant in the aqueous solution was 5 U / μl or more, aggregation occurred when the surfactant was not contained or when the concentration was low. From these results, it is suggested that the higher the concentration of the RNase inhibitor in the aqueous solution, the more likely it is that aggregation will occur, and it can be said that the occurrence of aggregation can be suppressed by adding a surfactant.
（実施例２）
界面活性剤の有無がＲＴ−ＰＣＲの反応系に与える影響の確認
（１）ＲＮＡサンプル
ＲＮＡサンプルとして、ＨｅＬａ細胞より精製したＴｏｔａｌ ＲＮＡを使用した。
（２）ＲＴ−ＰＣＲ反応
界面活性剤の反応系への影響の確認を行うためにＴｗｅｅｎ−２０を０．０１％含む４０Ｕ／μｌのＲＮａｓｅ阻害剤溶液を実施例１に記載の方法で調整し、ＲＴ−ＰＣＲ反応液の調整に使用した。反応液中に含まれるＴｗｅｅｎ−２０の終濃度は０．０００２５％となる。
ＲＴ−ＰＣＲは、以下のような組成及びサイクル条件により行った反応系の指標としてＰＣＲの反応効率、相関係数、Ｃｔ値を使用し、β−Ａｃｔｉｎを標的としたＲＴ−ＰＣＲを行った。
ＲｅｖｅｒＴｒａ Ａｃｅ（東洋紡） １ｕｎｉｔ、
ＲＮａｓｅ 阻害剤（東洋紡） １ｕｎｉｔ、
ｒＴｔｈ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（東洋紡） １ｕｎｉｔ、
抗Ｔｔｈ抗体 ０．４μｇ、
１０×Ｂｕｆｆｅｒ（東洋紡 ｒＴｔｈ ＤＮＡ ＰｏｌｙｍｅｒａｓｅのＢｕｆｆｅｒ）
１０ｍＭ ｄＮＴＰｓ（東洋紡） １．０μＬ、
５０μＭ β−ａｃｔｉｎフォアードプライマー（配列番号１） ０．３μＬ、
１００μＭ β−ａｃｔｉｎプローブ（配列番号２） ０．１μＬ、
５０μＭ β−ａｃｔｉｎリバースプライマー（配列番号３） ０．３μＬ、
ＲＮＡ ５μＬ、
血液 １０μＬ、
を含む反応液５０μＬを、以下の温度サイクルでＰＣＲ反応を行った。
５０℃ １０分
９５℃ １分
９５℃ １５秒−６０℃ ４５秒 ４５サイクル
（３）結果
その結果を表５に示す。
(Example 2)
Confirmation of the effect of the presence or absence of a surfactant on the reaction system of RT-PCR (1) RNA sample As an RNA sample, Total RNA purified from HeLa cells was used.
(2) In order to confirm the effect of the RT-PCR reaction surfactant on the reaction system, a 40 U / μl RNase inhibitor solution containing 0.01% of Tween-20 was prepared by the method described in Example 1. , Used to prepare RT-PCR reaction solution. The final concentration of Tween-20 contained in the reaction solution is 0.00025%.
For RT-PCR, RT-PCR targeting β-Actin was performed using PCR reaction efficiency, correlation coefficient, and Ct value as indicators of the reaction system performed under the following composition and cycle conditions.
RiverTra Ace (Toyobo) 1 unit,
RNase inhibitor (Toyobo) 1 unit,
rTth DNA Polymerase (Toyobo) 1 unit,
Anti-Tth antibody 0.4 μg,
10 x Buffer (Toyobo rTth DNA Polymerase Buffer)
10 mM dNTPs (Toyobo) 1.0 μL,
50 μM β-actin forward primer (SEQ ID NO: 1) 0.3 μL,
100 μM β-actin probe (SEQ ID NO: 2) 0.1 μL,
50 μM β-actin reverse primer (SEQ ID NO: 3) 0.3 μL,
RNA 5 μL,
10 μL of blood,
A PCR reaction was carried out with 50 μL of the reaction solution containing the above in the following temperature cycle.
50 ° C. 10 minutes 95 ° C. 1 minute 95 ° C. 15 seconds-60 ° C. 45 seconds 45 cycles (3) Results The results are shown in Table 5.
Ｔｗｅｅｎ−２０を添加した反応系と、していない反応系を比較した結果、Ｃｔ値、ＰＣＲ効率、相関係数に差は見られなかった。この結果によりＴｗｅｅｎ−２０によるＲＴ−ＰＣＲの阻害は見受けられないと言える。このほかにも、ノニデット Ｐ−４０、ＴｒｉｔｏｎＸ−１００、Ｂｒｉｇ３５、ＳＤＳ、ＣＨＡＰＳ、コール酸ナトリウム、デオキシコール酸ナトリウムでも同様に実験を実施し、ＲＴ−ＰＣＲの阻害は見受けらなかった。 As a result of comparing the reaction system to which Tween-20 was added and the reaction system to which Tween-20 was not added, no difference was observed in the Ct value, PCR efficiency, and correlation coefficient. From this result, it can be said that no inhibition of RT-PCR by Tween-20 is observed. In addition, the same experiments were carried out with Nonidet P-40, Triton X-100, Brig35, SDS, CHAPS, sodium cholic acid, and sodium deoxycholate, and no inhibition of RT-PCR was observed.
本発明は、ＲＮａｓｅ阻害剤を含む水溶液を使用または保存する際に水溶液中の凝集を抑制することができ、特に有用である。水溶液中のＲＮａｓｅ阻害剤の濃度が高いほど、凝集は発生しやすい。凝集の発生は作業上好ましくなく、ＲＮａｓｅ阻害剤を含む試薬の性能に影響を及ぼすため許容されない。本発明は界面活性剤を添加することで凝集を抑制することが可能であり、ＲＴ−ＰＣＲなどＲＮａｓｅ阻害剤を含む実験系において、阻害などの影響を与えないため、産業上も非常に有用である。 The present invention is particularly useful because it can suppress aggregation in an aqueous solution when using or storing an aqueous solution containing an RNase inhibitor. The higher the concentration of the RNase inhibitor in the aqueous solution, the more likely it is that aggregation will occur. Occurrence of agglomeration is unfavorable for work and is unacceptable as it affects the performance of reagents containing RNase inhibitors. The present invention is very useful in industry because it is possible to suppress aggregation by adding a surfactant and it does not have an influence such as inhibition in an experimental system containing an RNase inhibitor such as RT-PCR. is there.
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