JP5099455B2

JP5099455B2 - Method for selective amplification of RNA using emulsion

Info

Publication number: JP5099455B2
Application number: JP2009168285A
Authority: JP
Inventors: 哲也四方; 友亮松浦; 伯一市橋; 博也占部
Original assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Current assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Priority date: 2009-07-16
Filing date: 2009-07-16
Publication date: 2012-12-19
Anticipated expiration: 2029-07-16
Also published as: JP2011019461A

Description

本発明は、核酸増幅の技術分野に属する。より詳細には、本発明は、ＲＮＡ増幅の技術分野に属する。 The present invention belongs to the technical field of nucleic acid amplification. More particularly, the present invention belongs to the technical field of RNA amplification.

ＲＮＡ増幅法は、遺伝子操作や分子生物学の実験において重要であるのみならず、診断などにおいても重要な技術である。ＲＮＡ増幅を担う酵素として、Ｑβレプリカーゼが周知である。 The RNA amplification method is not only important in genetic manipulation and molecular biology experiments, but also an important technique in diagnosis and the like. Qβ replicase is well known as an enzyme responsible for RNA amplification.

Ｑβレプリカーゼは、大腸菌ファージＱβ（非特許文献１）のゲノムＲＮＡ（４２１７塩基長）を複製する、ＲＮＡ依存性ＲＮＡ複製酵素である（非特許文献２〜３）。Ｑβレプリカーゼは４量体で働くことが知られており、その構成は、ファージ由来のβサブユニットと宿主由来のＲｉｂｏｓｏｍａｌｐｒｏｔｅｉｎＳ１、伸張因子Ｔｕ（ＥＦ−Ｔｕ）およびＴｓ（ＥＦ−Ｔｓ）からなる（非特許文献４）。これらのうちで、活性に必須のサブユニットは、βサブユニット、ＥＦ−Ｔｕサブユニット、および、ＥＦ−Ｔｓサブユニットである。Ｓ１サブユニットは、特定の鋳型ＲＮＡ（例えば、ゲノムＲＮＡ）の複製に必須である。 Qβ replicase is an RNA-dependent RNA replication enzyme that replicates genomic RNA (4217 base length) of E. coli phage Qβ (Non-patent Document 1) (Non-patent Documents 2 to 3). Qβ replicase is known to work as a tetramer, and consists of a phage-derived β subunit and a host-derived Ribosomal protein S1, elongation factors Tu (EF-Tu) and Ts (EF-Ts). (Non-Patent Document 4). Among these, subunits essential for activity are β subunit, EF-Tu subunit, and EF-Ts subunit. The S1 subunit is essential for replication of specific template RNA (eg, genomic RNA).

Ｑβレプリカーゼは鋳型となるゲノムＲＮＡのプラス鎖から相補鎖のマイナス鎖を合成し、さらにそのマイナス鎖からプラス鎖を合成するといったサイクルで働き、その特徴から、鋳型ＲＮＡを指数的に増幅することが可能な酵素である（非特許文献５）。また、宿主因子であるＨｆｑによってその反応が促進されることが知られている（非特許文献６）。 Qβ replicase works in a cycle that synthesizes the minus strand of the complementary strand from the plus strand of the genomic RNA used as a template, and then synthesizes the plus strand from the minus strand. From its characteristics, it can amplify the template RNA exponentially. It is a possible enzyme (Non-patent Document 5). In addition, it is known that the reaction is promoted by Hfq which is a host factor (Non-patent Document 6).

しかしながら、試験管内でＱβレプリカーゼによるゲノムＲＮＡ複製反応を行った場合、ゲノムＲＮＡのような大きなＲＮＡはあまり複製されず、ｄｅｎｏｖｏ合成によって短いＲＮＡ（ｓｐｏｎｔａｎｅｏｕｓＲＮＡ（ｓｐＲＮＡ））が現れることが知られており、これが、ゲノムＲＮＡ複製反応の解析に大きな障害となっている。このｄｅｎｏｖｏ合成ＲＮＡの由来には様々な意見があり、例えば、反応液中のＮＴＰからの合成（非特許文献７〜８）、複製過程におけるＲＮＡの組み換えや（非特許文献９〜１１）、鋳型ＲＮＡの分解産物などが提唱されている。また、空気中からの混入なども挙げられており（非特許文献１２）、完全な除去や混入防止は不可能だと言われている。 However, when genomic RNA replication reaction with Qβ replicase is performed in vitro, large RNA such as genomic RNA is not replicated so much, and it is known that short RNA (spontaneous RNA (spRNA)) appears by de novo synthesis. This is a major obstacle to the analysis of the genomic RNA replication reaction. There are various opinions on the origin of this de novo synthetic RNA, for example, synthesis from NTP in the reaction solution (Non-patent Documents 7 to 8), recombination of RNA in the replication process (Non-patent Documents 9 to 11), A degradation product of template RNA has been proposed. Moreover, mixing from the air etc. is mentioned (nonpatent literature 12), and it is said that complete removal and mixing prevention are impossible.

Calendar, R. The bacteriophages, Edn. 2nd. (Oxford University Press,Oxford ; New York; 2006)Calendar, R. The bacteriophages, Edn. 2nd. (Oxford University Press, Oxford; New York; 2006) Haruna, I. & Spiegelman, S. Specific template requirements ofRNA replicases. Proc Natl Acad Sci U S A 54, 579-587 (1965)Haruna, I. & Spiegelman, S. Specific template requirements ofRNA replicases.Proc Natl Acad Sci U S A 54, 579-587 (1965) Chetverin, A.B. & Spirin, A.S. Replicable RNA vectors: prospectsfor cell-free gene amplification, expression, and cloning. Prog Nucleic AcidRes Mol Biol 51, 225-270 (1995)Chetverin, A.B. & Spirin, A.S.Replicable RNA vectors: prospectsfor cell-free gene amplification, expression, and cloning.Prog Nucleic AcidRes Mol Biol 51, 225-270 (1995) Brown, D. & Gold, L. RNA replication by Q beta replicase: a workingmodel. Proc NatlAcad Sci U S A 93, 11558-11562 (1996)Brown, D. & Gold, L. RNA replication by Q beta replicase: a workingmodel.Proc NatlAcad Sci U S A 93, 11558-11562 (1996) Biebricher, C.K., Eigen, M. & Luce, R. Kinetic analysis oftemplate-instructed and de novo RNA synthesis by Q beta replicase. J Mol Biol148, 391-410 (1981)Biebricher, C.K., Eigen, M. & Luce, R. Kinetic analysis of template-instructed and de novo RNA synthesis by Q beta replicase.J Mol Biol148, 391-410 (1981) Franze de Fernandez, M. T., Eoyang, L.. & August, J. T. Factorfraction required for the synthesis of bacteriophage Qβ RNA. Nature 219,588-590Franze de Fernandez, M. T., Eoyang, L .. & August, J. T. Factorfraction required for the synthesis of bacteriophage Qβ RNA.Nature 219,588-590

大きなＲＮＡの高効率複製のために、ＲＮＡ複製の障害となっているｓｐＲＮＡの発生を抑制する、新規のＲＮＡ複製法が求められている。本発明では、ＲＮＡ複製の障害となっているｓｐＲＮＡの発生を抑制する、新規のＲＮＡ複製法を提供することを、その課題とする。 For high-efficiency replication of large RNAs, there is a need for new RNA replication methods that suppress the generation of spRNAs that interfere with RNA replication. It is an object of the present invention to provide a novel RNA replication method that suppresses the generation of spRNA that is an obstacle to RNA replication.

本発明においては、エマルジョンを用いてＲＮＡ増幅溶液を微小区画化し、ｓｐＲＮＡを含む画分を大きなＲＮＡを増幅する画分と隔離し、その結果、従来法ではＱβレプリカーゼによって増幅することが不可能であった大きなＲＮＡの増幅が可能になったことを予想外に見出すことにより、上記課題を解決した。 In the present invention, the RNA amplification solution is micropartitioned using an emulsion, and the fraction containing spRNA is separated from the fraction that amplifies large RNA. As a result, the conventional method cannot be amplified by Qβ replicase. The above problem was solved by unexpectedly finding that a large RNA could be amplified.

従って、本発明は例えば以下を提供する。
（項目１）
以下：
（ａ）Ｑβレプリカーゼ；
（ｂ）反応緩衝液；および、
（ｃ）鋳型核酸；
を含有する水相成分、ならびに、油相成分を含む、核酸増幅のための油中水型エマルジョン。
（項目２）
項目１に記載の油中水型エマルジョンであって、
（ｉ）プライマー；および
（ｉｉ）固相支持体、
のいずれも含有しない、油中水型エマルジョン。
（項目３）
前記（ａ）Ｑβレプリカーゼが：
（ｃ１）
（ｃ１−１）配列番号２に示されるアミノ酸配列を含む、ポリペプチド、
（ｃ１−２）配列番号２に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含むポリペプチドであって、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、および、
（ｃ１−３）配列番号１に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドであって、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、
からなる群から選択される、ポリペプチド；
（ｃ２）
（ｃ２−１）配列番号９に示されるアミノ酸配列を含む、ポリペプチド、
（ｃ２−２）配列番号９に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含むポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、および、
（ｃ２−３）配列番号８に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、
からなる群から選択される、ポリペプチド；ならびに、
（ｃ３）
（ｃ３−１）配列番号１１に示されるアミノ酸配列を含む、ポリペプチド、
（ｃ３−２）配列番号１１に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含むポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチドおよび配列番号９に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、および、
（ｃ３−３）配列番号１０に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチドおよび配列番号９に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、
からなる群から選択される、ポリペプチド；
を含む、項目１に記載の油中水型エマルジョン。
（項目４）
前記（ａ）Ｑβレプリカーゼがさらに、
（ｃ４）
（ｃ４−１）配列番号１３に示されるアミノ酸配列を含む、ポリペプチド、
（ｃ４−２）配列番号１３に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含むポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチド、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合に、配列番号２に示されるアミノ酸配列からなるポリペプチド、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドの組み合わせよりも高いＲＮＡ複製活性を示す、ポリペプチド、および、
（ｃ４−３）配列番号１２に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチド、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合に、配列番号２に示されるアミノ酸配列からなるポリペプチド、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドの組み合わせよりも高いＲＮＡ複製活性を示す、ポリペプチド
からなる群から選択される、ポリペプチド
を含む、項目１に記載の油中水型エマルジョン。
（項目５）
前記（ｂ）反応緩衝液が、緩衝剤、塩、リボヌクレオチド、還元剤、および、タンパク質を含む水溶液である、項目１に記載の油中水型エマルジョン。
（項目６）
前記（ｃ）鋳型核酸が３’末端に核酸配列「ＣＣＣ」を有する、項目１に記載の油中水型エマルジョン。
（項目７）
前記（ｃ）鋳型核酸が、３００塩基以上の長さである、項目１に記載の油中水型エマルジョン。
（項目８）
項目１に記載の油中水型エマルジョンであって、直径が５μｍ以上２０μｍ以下のポアサイズを有するメンブレンを用いて製造されるか、または、ボルテックスの攪拌によって製造される、油中水型エマルジョン。
（項目９）
さらに、（ｄ）Ｈｆｑタンパク質を含む、項目１に記載の油中水型エマルジョンであって、ここで、該Ｈｆｑタンパク質が、
（ｄ１）配列番号４に示されるアミノ酸配列を含む、ポリペプチド；
（ｄ２）配列番号４に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含み、かつ、前記ＱβレプリカーゼのＲＮＡ複製活性を促進する、ポリペプチド；および、
（ｄ３）配列番号３に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされ、かつ、前記ＱβレプリカーゼのＲＮＡ複製活性を促進する、ポリペプチド；
からなる群から選択される、ポリペプチドである、項目１に記載の油中水型エマルジョン。
（項目１０）
さらに、（ｅ）乳化剤を含む、項目１に記載の油中水型エマルジョン。
（項目１１）
前記（ｅ）乳化剤が、ソルビタンモノオレエート、ポリオキシエチレンソルビタンモノオレエート、および、セチルジメチコンコポリオールからなる群から選択される、項目１０に記載の油中水型エマルジョン。
（項目１２）
項目１に記載の油中水型エマルジョンを含有する、ＲＮＡ複製のための組成物。
（項目１３）
核酸増幅反応のための油中水型エマルジョンの製造方法であって、以下の工程：
（１）（ａ）Ｑβレプリカーゼ、
（ｂ）反応緩衝液、
（ｃ）鋳型核酸、および、
（ｆ）油相成分、
を含有する混合物を調製する工程；ならびに、
（２）上記工程（１）の混合物を、メンブレンを通過させる工程、または、ボルテックスによって攪拌する工程、
を包含する、方法。
（項目１４）
項目１３に記載の油中水型エマルジョン製造方法であって、
前記工程（１）の混合物が、
（ｉ）プライマー；および
（ｉｉ）固相支持体、
のいずれも含有しない、方法。
（項目１５）
前記（ａ）Ｑβレプリカーゼが、
（ｃ１）
（ｃ１−１）配列番号２に示されるアミノ酸配列を含む、ポリペプチド、
（ｃ１−２）配列番号２に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含むポリペプチドであって、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、および、
（ｃ１−３）配列番号１に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドであって、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、
からなる群から選択される、ポリペプチド；
（ｃ２）
（ｃ２−１）配列番号９に示されるアミノ酸配列を含む、ポリペプチド、
（ｃ２−２）配列番号９に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含むポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、および、
（ｃ２−３）配列番号８に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、
からなる群から選択される、ポリペプチド；ならびに、
（ｃ３）
（ｃ３−１）配列番号１１に示されるアミノ酸配列を含む、ポリペプチド、
（ｃ３−２）配列番号１１に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含むポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチドおよび配列番号９に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、および、
（ｃ３−３）配列番号１０に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチドおよび配列番号９に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合にＲＮＡ複製活性を有する、ポリペプチド、
からなる群から選択される、ポリペプチド；
を含む、項目１３に記載の油中水型エマルジョン製造方法。
（項目１６）
前記（ａ）Ｑβレプリカーゼがさらに、
（ｃ４）
（ｃ４−１）配列番号１３に示されるアミノ酸配列を含む、ポリペプチド、
（ｃ４−２）配列番号１３に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含むポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチド、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合に、配列番号２に示されるアミノ酸配列からなるポリペプチド、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドの組み合わせよりも高いＲＮＡ複製活性を示す、ポリペプチド、および、
（ｃ４−３）配列番号１２に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドであって、配列番号２に示されるアミノ酸配列からなるポリペプチド、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドと組み合わせた場合に、配列番号２に示されるアミノ酸配列からなるポリペプチド、配列番号９に示されるアミノ酸配列からなるポリペプチドおよび配列番号１１に示されるアミノ酸配列からなるポリペプチドの組み合わせよりも高いＲＮＡ複製活性を示す、ポリペプチド
からなる群から選択される、ポリペプチド
を含む、項目１３に記載の油中水型エマルジョン製造方法。
（項目１７）
前記（ｂ）反応緩衝液が、緩衝剤、塩、リボヌクレオチド、還元剤、および、タンパク質を含む水溶液である、項目１３に記載の油中水型エマルジョン製造方法。
（項目１８）
前記（ｃ）鋳型核酸が３’末端に核酸配列「ＣＣＣ」を有する核酸である、項目１３に記載の油中水型エマルジョン製造方法。
（項目１９）
前記（ｃ）鋳型核酸が、３００塩基以上の長さである、項目１３に記載の油中水型エマルジョン製造方法。
（項目２０）
前記メンブレンのポアサイズの直径が５μｍ以上２０μｍ以下である、項目１３に記載の油中水型エマルジョン製造方法。
（項目２１）
前記工程（１）の混合物がさらに（ｄ）Ｈｆｑタンパク質を含む、項目１３に記載の油中水型エマルジョン製造方法であって、ここで、該Ｈｆｑタンパク質が、
（ｄ１）配列番号４に示されるアミノ酸配列を含む、ポリペプチド；
（ｄ２）配列番号４に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含み、かつ、前記ＱβレプリカーゼのＲＮＡ複製活性を促進する、ポリペプチド；および、
（ｄ３）配列番号３に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされ、かつ、前記ＱβレプリカーゼのＲＮＡ複製活性を促進する、ポリペプチド；
からなる群から選択される、ポリペプチドである、油中水型エマルジョン製造方法。
（項目２２）
前記工程（１）の混合物がさらに（ｅ）乳化剤を含む、項目１３に記載の油中水型エマルジョン製造方法。
（項目２３）
前記（ｅ）乳化剤が、ソルビタンモノオレエート、ポリオキシエチレンソルビタンモノオレエート、および、セチルジメチコンコポリオールからなる群から選択される、項目２２に記載の油中水型エマルジョン製造方法。 Accordingly, the present invention provides, for example:
(Item 1)
Less than:
(A) Qβ replicase;
(B) a reaction buffer; and
(C) a template nucleic acid;
A water-in-oil emulsion for nucleic acid amplification, comprising an aqueous phase component containing an oil phase, and an oil phase component.
(Item 2)
A water-in-oil emulsion according to item 1,
(I) a primer; and (ii) a solid support.
A water-in-oil emulsion that does not contain any of the above.
(Item 3)
The (a) Qβ replicase is:
(C1)
(C1-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2,
(C1-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 2 and consisting of the amino acid sequence shown in SEQ ID NO: 9 A polypeptide having RNA replication activity when combined with the polypeptide and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C1-3) A polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 1, comprising the amino acid sequence shown in SEQ ID NO: 9 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide comprising the amino acid sequence shown in FIG.
A polypeptide selected from the group consisting of:
(C2)
(C2-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 9,
(C2-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 9, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having RNA replication activity when combined with the polypeptide and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C2-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 8, comprising the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide comprising the amino acid sequence shown in FIG.
A polypeptide selected from the group consisting of:
(C3)
(C3-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11,
(C3-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 11, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having RNA replication activity when combined with the polypeptide and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, and
(C3-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 10, and comprising the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide consisting of the amino acid sequence shown in 9,
A polypeptide selected from the group consisting of:
The water-in-oil emulsion according to item 1, comprising
(Item 4)
The (a) Qβ replicase is further
(C4)
(C4-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 13,
(C4-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 13, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 when combined with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11; A polypeptide having an RNA replication activity higher than the combination of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C4-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 12, comprising the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 when combined with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, amino acid sequence shown in SEQ ID NO: 9 The oil according to item 1, comprising a polypeptide selected from the group consisting of polypeptides that exhibit higher RNA replication activity than a combination of a polypeptide consisting of and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11. Water emulsion.
(Item 5)
Item 2. The water-in-oil emulsion according to Item 1, wherein the reaction buffer (b) is an aqueous solution containing a buffer, a salt, a ribonucleotide, a reducing agent, and a protein.
(Item 6)
Item 2. The water-in-oil emulsion according to Item 1, wherein the template nucleic acid has the nucleic acid sequence “CCC” at the 3 ′ end.
(Item 7)
Item 2. The water-in-oil emulsion according to Item 1, wherein the template nucleic acid has a length of 300 bases or more.
(Item 8)
2. The water-in-oil emulsion according to item 1, wherein the water-in-oil emulsion is produced using a membrane having a pore size of 5 μm or more and 20 μm or less, or produced by vortexing.
(Item 9)
The water-in-oil emulsion according to item 1, further comprising (d) Hfq protein, wherein the Hfq protein is
(D1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4;
(D2) a polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 4 and promoting RNA replication activity of the Qβ replicase; and ,
(D3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 3 and that promotes the RNA replication activity of the Qβ replicase;
The water-in-oil emulsion according to item 1, which is a polypeptide selected from the group consisting of:
(Item 10)
The water-in-oil emulsion according to item 1, further comprising (e) an emulsifier.
(Item 11)
Item 11. The water-in-oil emulsion according to Item 10, wherein the (e) emulsifier is selected from the group consisting of sorbitan monooleate, polyoxyethylene sorbitan monooleate, and cetyl dimethicone copolyol.
(Item 12)
A composition for RNA replication, comprising the water-in-oil emulsion according to item 1.
(Item 13)
A method for producing a water-in-oil emulsion for nucleic acid amplification reaction comprising the following steps:
(1) (a) Qβ replicase,
(B) a reaction buffer,
(C) a template nucleic acid, and
(F) oil phase component,
Preparing a mixture containing: and
(2) The step of passing the mixture of the above step (1) through a membrane or the step of stirring by vortex,
Including the method.
(Item 14)
A method for producing a water-in-oil emulsion according to item 13, comprising:
The mixture of step (1) is
(I) a primer; and (ii) a solid support.
None of the methods.
(Item 15)
The (a) Qβ replicase is
(C1)
(C1-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2,
(C1-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 2 and consisting of the amino acid sequence shown in SEQ ID NO: 9 A polypeptide having RNA replication activity when combined with the polypeptide and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C1-3) A polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 1, comprising the amino acid sequence shown in SEQ ID NO: 9 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide comprising the amino acid sequence shown in FIG.
A polypeptide selected from the group consisting of:
(C2)
(C2-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 9,
(C2-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 9, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having RNA replication activity when combined with the polypeptide and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C2-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 8, comprising the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide comprising the amino acid sequence shown in FIG.
A polypeptide selected from the group consisting of:
(C3)
(C3-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11,
(C3-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 11, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having RNA replication activity when combined with the polypeptide and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, and
(C3-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 10, and comprising the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide consisting of the amino acid sequence shown in 9,
A polypeptide selected from the group consisting of:
14. The method for producing a water-in-oil emulsion according to item 13, comprising:
(Item 16)
The (a) Qβ replicase is further
(C4)
(C4-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 13,
(C4-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 13, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 when combined with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11; A polypeptide having an RNA replication activity higher than the combination of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C4-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 12, comprising the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 when combined with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, amino acid sequence shown in SEQ ID NO: 9 14. In oil according to item 13, comprising a polypeptide selected from the group consisting of polypeptides that exhibit higher RNA replication activity than a combination of a polypeptide consisting of and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11. Water emulsion production method.
(Item 17)
Item 14. The method for producing a water-in-oil emulsion according to Item 13, wherein the reaction buffer (b) is an aqueous solution containing a buffer, a salt, a ribonucleotide, a reducing agent, and a protein.
(Item 18)
Item 14. The method for producing a water-in-oil emulsion according to Item 13, wherein the template nucleic acid is a nucleic acid having a nucleic acid sequence “CCC” at the 3 ′ end.
(Item 19)
Item 14. The method for producing a water-in-oil emulsion according to Item 13, wherein the template nucleic acid has a length of 300 bases or more.
(Item 20)
Item 14. The method for producing a water-in-oil emulsion according to Item 13, wherein the pore size diameter of the membrane is 5 µm or more and 20 µm or less.
(Item 21)
14. The method for producing a water-in-oil emulsion according to item 13, wherein the mixture of step (1) further comprises (d) Hfq protein, wherein the Hfq protein is
(D1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4;
(D2) a polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 4 and promoting RNA replication activity of the Qβ replicase; and ,
(D3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 3 and that promotes the RNA replication activity of the Qβ replicase;
A method for producing a water-in-oil emulsion, which is a polypeptide selected from the group consisting of:
(Item 22)
Item 14. The method for producing a water-in-oil emulsion according to Item 13, wherein the mixture of the step (1) further comprises (e) an emulsifier.
(Item 23)
Item 23. The method for producing a water-in-oil emulsion according to Item 22, wherein the (e) emulsifier is selected from the group consisting of sorbitan monooleate, polyoxyethylene sorbitan monooleate, and cetyl dimethicone copolyol.

本発明は、従来法ではＱβレプリカーゼによって増幅不可能であるような大きなＲＮＡの増幅を可能にするという効果を奏する。さらに、本発明においては、Ｑβレプリカーゼ活性がｓｐＲＮＡの阻害を受けないため、従来法では不可能であったＱβレプリカーゼの速度論的解析が可能になるという効果もまた、奏される。 The present invention has the effect of enabling amplification of large RNAs that cannot be amplified by Qβ replicase in the conventional method. Furthermore, in the present invention, since the Qβ replicase activity is not inhibited by spRNA, the effect of enabling kinetic analysis of Qβ replicase, which was impossible with the conventional method, is also achieved.

図１は、Ｓ２２２ＲＮＡを、試験管内（◆）、ボルテックスを用いて調製した油中水型エマルジョン（「Ｗ／Ｏエマルジョン」ともいう）内（○）、および、メンブレンを用いて調製したＷ／Ｏエマルジョン（●）を用いて増幅した結果を示す。FIG. 1 shows S222 RNA in a test tube (♦), a water-in-oil emulsion prepared using a vortex (also referred to as “W / O emulsion”) (◯), and a W / W prepared using a membrane. The results of amplification using O emulsion (●) are shown. 図２は、ＲＮＡ増幅反応液を調製した試験管内のβサブユニット量（「試験管」）、親水性メンブレンを用いて調製したＷ／Ｏエマルジョン内のβサブユニット量（「親水性」）、疎水性メンブレンを用いて調製したＷ／Ｏエマルジョン内のβサブユニット量（「疎水性」）、および、ボルテックスを用いて調製したＷ／Ｏエマルジョン内のβサブユニット量（「ボルテックス」）を、試料の調製に用いたβサブユニット（「コントロール」）の相対値として示したグラフである。調製時にＤＴＴを用いた場合は、（「＋」）を記載し、ＤＴＴを用いなかった場合は、（「−」）を記載した。FIG. 2 shows the amount of β subunit in the test tube prepared with the RNA amplification reaction solution (“test tube”), the amount of β subunit in the W / O emulsion prepared using the hydrophilic membrane (“hydrophilic”), The amount of β subunit in the W / O emulsion prepared using the hydrophobic membrane (“hydrophobic”) and the amount of β subunit in the W / O emulsion prepared using vortex (“vortex”) It is the graph shown as a relative value of (beta) subunit ("control") used for preparation of a sample. When DTT was used at the time of preparation, ("+") was described, and when DTT was not used, ("-") was described. 図３は、試験管内の反応（「試験管」）、親水性メンブレンを用いて調製したＷ／Ｏエマルジョン内のＲＮＡ増幅反応（「親水性」）、疎水性メンブレンを用いて調製したＷ／Ｏエマルジョン内のＲＮＡ増幅反応（「疎水性」）、および、ボルテックスを用いて調製したＷ／Ｏエマルジョン内のＲＮＡ増幅反応（「ボルテックス」）を比較したグラフである。調製時にＤＴＴを用いた場合は、（「＋」）を記載し、ＤＴＴを用いなかった場合は、（「−」）を記載した。FIG. 3 shows reaction in a test tube (“test tube”), RNA amplification reaction in a W / O emulsion prepared using a hydrophilic membrane (“hydrophilic”), and W / O prepared using a hydrophobic membrane. FIG. 5 is a graph comparing RNA amplification reaction in emulsion (“hydrophobic”) and RNA amplification reaction in W / O emulsion prepared using vortex (“vortex”). When DTT was used at the time of preparation, ("+") was described, and when DTT was not used, ("-") was described. 図４は、種々の濃度のＨｆｑを用いた場合のＲＮＡ複製反応の結果を示すグラフである。FIG. 4 is a graph showing the results of RNA replication reaction when various concentrations of Hfq are used. 図５は、Ｑβレプリカーゼ２によるＲＮＡ増幅反応の指数期における経時的変化を示すグラフである。FIG. 5 is a graph showing the change over time in the exponential phase of the RNA amplification reaction by Qβ replicase 2. 図６は、Ｑβレプリカーゼ２によるＲＮＡ増幅反応の線形期における経時的変化を示すグラフである。FIG. 6 is a graph showing the change over time in the linear phase of the RNA amplification reaction by Qβ replicase 2. 図７は、Ｑβレプリカーゼ２によって増幅されたＲＮＡ中の＋鎖と−鎖を、経時的に測定した結果である。FIG. 7 shows the results of measuring the + and − strands in RNA amplified by Qβ replicase 2 over time. 図８は、アニーリング実験の結果の電気泳動写真である。FIG. 8 is an electrophoretogram of the results of the annealing experiment. 図９は、指数増幅条件（●）および線形増幅条件（○）における各時点での２本鎖率を算出した結果である。FIG. 9 shows the results of calculating the double-stranded ratio at each time point under the exponential amplification condition (●) and the linear amplification condition (◯).

以下、本発明を説明する。本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念も含むことが理解されるべきである。また、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。 The present invention will be described below. Throughout this specification, it should be understood that expression in the singular includes the concept of the plural unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

以下に本明細書において特に使用される用語の定義を列挙する。 Listed below are definitions of terms particularly used in the present specification.

本発明において使用する場合、用語「Ｑβレプリカーゼ」とは、ＱβファージのＲＮＡ複製酵素およびその改変体をいう。ＲＮＡ複製活性を保持する限り、あらゆる改変体が、本発明の「Ｑβレプリカーゼ」に包含される。例えば、本発明のＱβレプリカーゼとしては、（１）βサブユニット（例えば、配列番号１の核酸配列によってコードされる配列番号２のアミノ酸配列からなるポリペプチド）、または、Ｑβレプリカーゼにアセンブルした場合にＲＮＡ複製活性を保持するβサブユニットの改変体、（２）ＥＦ−Ｔｕサブユニット（例えば、配列番号１０の核酸配列によってコードされる配列番号１１のアミノ酸配列からなるポリペプチド）、または、Ｑβレプリカーゼにアセンブルした場合にＲＮＡ複製活性を保持するＥＦ−Ｔｕサブユニットの改変体、（３）ＥＦ−Ｔｓサブユニット（例えば、配列番号８の核酸配列によってコードされる配列番号９のアミノ酸配列からなるポリペプチド）、または、Ｑβレプリカーゼにアセンブルした場合にＲＮＡ複製活性を保持するＥＦ−Ｔｓサブユニットの改変体を含むタンパク質が挙げられるが、これに限定されない。また、必要に応じて、Ｑβレプリカーゼは、（４）Ｓ１サブユニット（例えば、配列番号１２の核酸配列によってコードされる配列番号１３のアミノ酸配列からなるポリペプチド）、または、Ｑβレプリカーゼにアセンブルした場合にＲＮＡ複製活性を促進するＳ１サブユニットの改変体を含んでもよい。サブユニットの改変体としては、野生型ポリペプチドのアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含みむポリペプチド、および、野生型ポリペプチドをコードする核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされるポリペプチドが挙げられるが、これらに限定されない。 As used in the present invention, the term “Qβ replicase” refers to an RNA replicase enzyme of Qβ phage and a variant thereof. Any variants are included in the “Qβ replicase” of the present invention as long as they retain RNA replication activity. For example, the Qβ replicase of the present invention includes (1) a β subunit (for example, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 encoded by the nucleic acid sequence of SEQ ID NO: 1), or when assembled into a Qβ replicase. A variant of a β subunit that retains RNA replication activity, (2) an EF-Tu subunit (eg, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 11 encoded by the nucleic acid sequence of SEQ ID NO: 10), or a Qβ replicase A modified EF-Tu subunit that retains RNA replication activity when assembled into (3) an EF-Ts subunit (for example, a poly-nucleotide comprising the amino acid sequence of SEQ ID NO: 9 encoded by the nucleic acid sequence of SEQ ID NO: 8) Peptide) or RNA replication activity when assembled into Qβ replicase Proteins comprising variants of EF-Ts subunit for holding include, but are not limited thereto. Further, if necessary, the Qβ replicase is assembled into (4) S1 subunit (for example, a polypeptide comprising the amino acid sequence of SEQ ID NO: 13 encoded by the nucleic acid sequence of SEQ ID NO: 12) or Qβ replicase May contain a variant of the S1 subunit that promotes RNA replication activity. Variants of subunits include polypeptides that include an amino acid sequence that includes one or several deletions, additions, or substitutions in the amino acid sequence of the wild-type polypeptide, and nucleic acids that encode the wild-type polypeptide Examples include, but are not limited to, a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to a sequence.

本発明において使用する場合、用語「反応緩衝液」とは、本来ＲＮＡ複製活性を有する本発明のＱβレプリカーゼが、そのＲＮＡ複製活性を発揮することができる環境を提供する溶液をいう。反応緩衝液は、緩衝剤、塩、および、リボヌクレオチドを含む水溶液であるが、プライマーや固相支持体は含まない。好ましくは、反応緩衝液は、還元剤、および、タンパク質を含む。緩衝剤としては、当該分野において周知の緩衝剤であってＱβレプリカーゼの活性を阻害しない緩衝剤を用いることができる。緩衝剤としては、例えば、Ｔｒｉｓ、および、Ｈｅｐｅｓが挙げられるが、これに限定されない。塩としては、当該分野において周知の塩であってＱβレプリカーゼの活性を阻害しない塩を用いることができる。塩としては、例えば、ＭｇＣｌ_２、および、酢酸マグネシウムが挙げられるが、これに限定されない。使用する塩濃度は、５〜２０ｍＭ、好ましくは５〜１０ｍＭ、最も好ましくは約５ｍＭであるが、これらに限定されない。リボヌクレオチドは、Ｑβレプリカーゼの基質である。リボヌクレオチドとしては、代表的には、アデノシン三リン酸（ＡＴＰ）、グアノシン三リン酸（ＧＴＰ）、ウリジン三リン酸（ＵＴＰ）、および、シチジン三リン酸（ＣＴＰ）が挙げられるが、これに限定されない。これらリボヌクレオチドの修飾物であって、Ｑβレプリカーゼの基質となるリボヌクレオチドもまた、本発明のリボヌクレオチドに包含される。タンパク質としては、当該分野において周知のタンパク質であってＱβレプリカーゼの活性を阻害しないタンパク質を、Ｑβレプリカーゼの安定化のために用いることができる。タンパク質としては、例えば、ＢＳＡ（ウシ血清アルブミン）が挙げられるが、これに限定されない。還元剤としては、当該分野において周知の還元剤であってＱβレプリカーゼの活性を阻害しない還元剤を用いることができる。還元剤としては、例えば、ＤＴＴ（ジチオスレイトール）、および、βメルカプトエタノールが挙げられるが、これに限定されない。 As used in the present invention, the term “reaction buffer” refers to a solution that provides an environment in which the Qβ replicase of the present invention, which originally has RNA replication activity, can exhibit its RNA replication activity. The reaction buffer is an aqueous solution containing a buffer, a salt, and ribonucleotide, but does not contain a primer or a solid support. Preferably, the reaction buffer contains a reducing agent and a protein. As the buffer, a buffer that is well-known in the art and does not inhibit the activity of Qβ replicase can be used. Examples of the buffer include, but are not limited to, Tris and Hepes. As the salt, a salt well known in the art and not inhibiting the activity of Qβ replicase can be used. Examples of the salt include, but are not limited to, MgCl ₂ and magnesium acetate. The salt concentration used is 5-20 mM, preferably 5-10 mM, most preferably about 5 mM, but is not limited thereto. Ribonucleotides are substrates for Qβ replicase. Ribonucleotides typically include adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and cytidine triphosphate (CTP). It is not limited. Ribonucleotides that are modified products of these ribonucleotides and serve as substrates for Qβ replicase are also included in the ribonucleotides of the present invention. As the protein, a protein that is well-known in the art and does not inhibit the activity of Qβ replicase can be used for the stabilization of Qβ replicase. Examples of the protein include, but are not limited to, BSA (bovine serum albumin). As the reducing agent, a reducing agent that is well-known in the art and does not inhibit the activity of Qβ replicase can be used. Examples of the reducing agent include, but are not limited to, DTT (dithiothreitol) and β-mercaptoethanol.

本発明において使用する場合、用語「プライマー」とは、数塩基長〜数百塩基長の核酸をいう。 As used in the present invention, the term “primer” refers to a nucleic acid having a length of several bases to several hundred bases.

本発明において使用する場合、用語「固相支持体」とは、水性溶媒に不溶性の固体をいう。 As used herein, the term “solid phase support” refers to a solid that is insoluble in an aqueous solvent.

本発明において使用する場合、用語「鋳型核酸」とは、本発明の方法によって増幅されるＲＮＡをいう。鋳型核酸は、好ましくは３００塩基以上、より好ましくは６００塩基以上、さらにより好ましくは１２００塩基以上、最も好ましくは４０００塩基以上であるがこれらに限定されない。本発明の鋳型核酸は、Ｑβレプリカーゼの基質となるために、好ましくは、３’末端に核酸配列「ＣＣＣ」を有する。 As used in the present invention, the term “template nucleic acid” refers to RNA amplified by the method of the present invention. The template nucleic acid is preferably 300 bases or more, more preferably 600 bases or more, even more preferably 1200 bases or more, and most preferably 4000 bases or more, but is not limited thereto. The template nucleic acid of the present invention preferably has a nucleic acid sequence “CCC” at the 3 ′ end in order to serve as a substrate for Qβ replicase.

本発明において使用する場合、用語「エマルジョン」とは、水相成分と油相成分という極性の異なる成分を混合した分散系溶液をいう。油相成分としては、例えば、ミネラルオイル、デカン、および、流動パラフィンが挙げられるが、これに限定されない。本発明のエマルジョンは、好ましくは、油中水型エマルジョン（「Ｗ／Ｏエマルジョン」ともいう）であり、また、好ましくは、乳化剤を含む。 As used in the present invention, the term “emulsion” refers to a dispersion solution in which components having different polarities such as an aqueous phase component and an oil phase component are mixed. Examples of the oil phase component include mineral oil, decane, and liquid paraffin, but are not limited thereto. The emulsion of the present invention is preferably a water-in-oil emulsion (also referred to as “W / O emulsion”), and preferably contains an emulsifier.

本発明において使用する場合、用語「乳化剤」とは、両親媒性の物質であり、本発明のエマルジョンを安定化する物質をいう。本発明の乳化剤としては、例えば、ソルビタンモノオレエート、ポリオキシエチレンソルビタンモノオレエート、および、セチルジメチコンコポリオールが挙げられるが、これに限定されない。 As used in the present invention, the term “emulsifier” refers to a substance that is amphiphilic and stabilizes the emulsion of the present invention. Examples of the emulsifier of the present invention include, but are not limited to, sorbitan monooleate, polyoxyethylene sorbitan monooleate, and cetyl dimethicone copolyol.

本発明において使用する場合、用語「Ｈｆｑタンパク質」とは、Ｑβファージの宿主細胞（例えば、Escherichia coli）のタンパク質であって、ＱβレプリカーゼのＲＮＡ複製活性を促進するタンパク質をいう。例えば、本発明のＨｆｑタンパク質としては、（ｄ１）配列番号４に示されるアミノ酸配列を含む、ポリペプチド、（ｄ２）配列番号４に示されるアミノ酸配列に、１または数個の欠失、付加、または、置換を含むアミノ酸配列を含み、かつ、前記ＱβレプリカーゼのＲＮＡ複製活性を促進する、ポリペプチド、および、（ｄ３）配列番号３に示される核酸配列に対してストリンジェントな条件でハイブリダイズする核酸によってコードされ、かつ、前記ＱβレプリカーゼのＲＮＡ複製活性を促進するポリペプチド、からなる群から選択される、ポリペプチドが挙げられるが、これらに限定されない。 As used in the present invention, the term “Hfq protein” refers to a protein of a Qβ phage host cell (eg, Escherichia coli) that promotes the RNA replication activity of Qβ replicase. For example, the Hfq protein of the present invention includes (d1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4, (d2) one or several deletions, additions to the amino acid sequence represented by SEQ ID NO: 4, Alternatively, it hybridizes under stringent conditions to a polypeptide comprising an amino acid sequence containing a substitution and promoting the RNA replication activity of the Qβ replicase, and (d3) the nucleic acid sequence shown in SEQ ID NO: 3. Examples include, but are not limited to, a polypeptide selected from the group consisting of a polypeptide encoded by a nucleic acid and promoting the RNA replication activity of the Qβ replicase.

（エマルジョンの調製）
本発明のエマルジョン（油中水エマルジョン）は、好ましくは乳化剤を含む。例えば、本発明のエマルジョンは、乳化剤を適当濃度加えた油相成分に少量の水相成分を加え攪拌することにより調製することができる。本発明のエマルジョンでは、好ましくは、油相成分中の水相成分は均一な球形になる。エマルジョンの調製方法としては、種々の方法が周知である。ボルテックスのせん断力による拡散法では、例えば、油相成分４８０μＬに対して水相成分２０μＬを添加し、４℃ ３２００ｒｐｍ（例えば、VORTEX-GENIE2（Scientific Industries）の場合、機械目盛り８）で３０秒間攪拌することによりエマルジョンを調製するが、この方法に限定されることはなく、当業者は、種々の周知の方法を用いることができる。メンブレンを用いる場合、例えば、油相成分９６０μＬに対して水相成分４０μＬを添加し、４℃でメンブレンを２０往復通過させるという方法を用いることができるが、この方法に限定されることなく、当業者は、種々の周知の方法を用いることができる。また、本発明において使用するメンブレンのポアサイズは、５μｍ以上２０μｍ以下であり、好ましくは２０μｍであるが、これらに限定されない。マイクロ流路を用いる場合、例えば、十字型の流路を用い、上から水層、左右から油層を流すことにより、下の流路からエマルションを回収できるが、この方法に限定されることなく、当業者は、種々の周知の方法を用いることができる。ソニケーションを用いる場合、例えば、プローブ型のソニケーターの場合、プローブを油層と水層の混じった溶液中に挿入し、出力するという方法を用いることができるが、この方法に限定されることなく、当業者は、種々の周知の方法を用いることができる。 (Preparation of emulsion)
The emulsion of the present invention (water-in-oil emulsion) preferably contains an emulsifier. For example, the emulsion of the present invention can be prepared by adding a small amount of an aqueous phase component to an oil phase component to which an appropriate concentration of an emulsifier is added and stirring. In the emulsion of the present invention, the aqueous phase component in the oil phase component is preferably in a uniform spherical shape. As a method for preparing the emulsion, various methods are well known. In the diffusion method using a vortex shear force, for example, 20 μL of an aqueous phase component is added to 480 μL of an oil phase component, and stirred for 30 seconds at 3200 rpm at 4 ° C. (for example, mechanical scale 8 in the case of VORTEX-GENIE2 (Scientific Industries)). However, the present invention is not limited to this method, and those skilled in the art can use various well-known methods. In the case of using a membrane, for example, a method of adding 40 μL of an aqueous phase component to 960 μL of an oil phase component and passing the membrane 20 times at 4 ° C. can be used, but the method is not limited to this method. A person skilled in the art can use various well-known methods. The pore size of the membrane used in the present invention is 5 μm or more and 20 μm or less, preferably 20 μm, but is not limited thereto. When using a micro-channel, for example, using a cross-shaped channel, the emulsion can be recovered from the lower channel by flowing the water layer from the top and the oil layer from the left and right, but without being limited to this method, Those skilled in the art can use various well-known methods. When using sonication, for example, in the case of a probe-type sonicator, a method of inserting and outputting a probe into a solution in which an oil layer and an aqueous layer are mixed can be used, but the method is not limited to this method. Those skilled in the art can use various well-known methods.

（核酸増幅のための油中水型エマルジョンの調製）
本発明の一つの局面において、（ａ）Ｑβレプリカーゼ、（ｂ）反応緩衝液、および、（ｃ）鋳型核酸を含有する水相成分、ならびに、油相成分を含む、核酸増幅のための油中水型エマルジョンが提供される。本発明の油中水型エマルジョンは、好ましくは、プライマーも固相支持体も含有しない。鋳型核酸は、好ましくは、ＲＮＡである。このＲＮＡを鋳型とし、反応緩衝液中のヌクレオチドを基質として、Ｑβレプリカーゼは、ＲＮＡの増幅反応を行う。 (Preparation of water-in-oil emulsion for nucleic acid amplification)
In one aspect of the present invention, in oil for nucleic acid amplification, comprising (a) Qβ replicase, (b) a reaction buffer, and (c) an aqueous phase component containing a template nucleic acid, and an oil phase component A water emulsion is provided. The water-in-oil emulsion of the present invention preferably contains no primer or solid support. The template nucleic acid is preferably RNA. Using this RNA as a template and nucleotides in a reaction buffer as a substrate, Qβ replicase performs an amplification reaction of RNA.

本発明の別の局面において、核酸増幅反応のための油中水型エマルジョンの製造方法であって、以下の工程：
（１）（ａ）Ｑβレプリカーゼ、
（ｂ）反応緩衝液、
（ｃ）鋳型核酸、および、
（ｆ）油相成分、
を含有する混合物を調製する工程；ならびに、
（２）上記工程（１）の混合物から油中水型エマルジョンを調製する工程、
を包含する方法が提供される。本発明の油中水型エマルジョンは、好ましくは、プライマーも固相支持体も含有しない。鋳型核酸は、好ましくは、ＲＮＡである。上記方法において製造された油中水型エマルジョンでは、このＲＮＡを鋳型とし、反応緩衝液中のヌクレオチドを基質として、Ｑβレプリカーゼが、ＲＮＡの増幅反応を行う。 In another aspect of the present invention, a method for producing a water-in-oil emulsion for nucleic acid amplification reaction, comprising the following steps:
(1) (a) Qβ replicase,
(B) a reaction buffer,
(C) a template nucleic acid, and
(F) oil phase component,
Preparing a mixture containing: and
(2) preparing a water-in-oil emulsion from the mixture of the above step (1),
Is provided. The water-in-oil emulsion of the present invention preferably contains no primer or solid support. The template nucleic acid is preferably RNA. In the water-in-oil emulsion produced in the above method, Qβ replicase performs RNA amplification reaction using this RNA as a template and nucleotides in a reaction buffer as a substrate.

（材料および方法）
１．鋳型ＲＮＡの調製
１．１．インビボ抽出ゲノムの調整（インビボＲＮＡ）の調製
鋳型ゲノムＲＮＡとして、Ａ２タンパク、コートタンパク、βサブユニットをコードする約４２１７塩基長のＲＮＡを用いた。大腸菌Ａ／λを５ｍＬＬＢ培地で３７℃ １２０ｒｐｍでオーバーナイト前培養し、その５０μＬを５ｍＬＬＢ培地でＯＤ（６００ｎｍ）が０．１〜０．２になるまで培養した（大腸菌Ａ／λ：１．０×１０^８ｃｆｕ／ｍＬ）。次に１ＭＣａＣｌ_２を終濃度１０ｍＭになるように添加し、ファージタイターが１．０×１０^１１ｐｆｕ／ｍＬになるようにＱβファージを加え、ＭＯＩ（ＭｕｌｔｉｐｌｉｃｉｔｙｏｆＩｎｆｅｃｔｉｏｎ）が０．０１になるまで約５時間振とう培養した。次にクロロホルム１２５ｕｌ加え２〜３分間振とう培養し、５０００ｇで２０分間遠心分離を行い、上清を回収した（４回）。回収溶液をポアサイズ０．２２μｍのフィルターで濾過を行った。次に等量のフェノール／クロロホルムを素早く混和し、２５℃ １００００ｒｐｍで１０分間遠心分離を行い、上清を回収した（３回）。回収溶液に３Ｍ酢酸ナトリウム（ｐＨ５．２）を４０μＬ加え、さらにイソプロパノールを４４０μＬ加え素早く混和し、−８０℃で１５分間静置した。次に、その溶液を４℃ １４０００ｒｐｍで１０分間遠心分離を行い、上清をピペッティング除去し、７０％エタノールを４００μＬ加え、さらに４℃ １４０００ｒｐｍで５分間遠心分離を行い、上清をピペッティング除去した。残存エタノールをデシケーションした後に、ＲＮａｓｅＦｒｅｅＷａｔｅｒを４００μＬ加え穏やかに混和、さらに５ＭＬｉＣｌを４００μＬ加え穏やかに混和した。４℃で２時間静置後、４℃ １２０００ｒｐｍで１５分間遠心分離を行い、上清をピペッティング除去した。さらにもう一度、７０％エタノールを４００μＬ加え、４℃ １４０００ｒｐｍで５分間遠心分離を行い、上清をピペッティング除去した後、残存エタノールをデシケーションし、ＲＮａｓｅＦｒｅｅＷａｔｅｒを３０μＬ加えた。産物をＲＮＡＭｉｎｉｅｌｕｔｅで精製し目的のゲノムＲＮＡを得た。 (Materials and methods)
1. Preparation of template RNA 1.1. Preparation of In Vivo Extracted Genome Preparation (In Vivo RNA) As a template genomic RNA, RNA having a length of about 4217 bases encoding A2 protein, coat protein and β subunit was used. E. coli A / λ was precultured overnight in 5 mL LB medium at 37 ° C. and 120 rpm, and 50 μL thereof was cultured in 5 mL LB medium until OD (600 nm) became 0.1 to 0.2 (E. coli A / λ: 1 0.0 × 10 ⁸ cfu / mL). Next, 1M CaCl ₂ is added to a final concentration of 10 mM, Qβ phage is added so that the phage titer is 1.0 × 10 ¹¹ pfu / mL, and MOI (Multiplicity of Infection) is 0.01. Cultured with shaking for about 5 hours. Next, 125 ul of chloroform was added and cultured with shaking for 2 to 3 minutes, followed by centrifugation at 5000 g for 20 minutes, and the supernatant was collected (4 times). The recovered solution was filtered through a filter having a pore size of 0.22 μm. Next, an equal amount of phenol / chloroform was quickly mixed and centrifuged at 25 ° C. and 10,000 rpm for 10 minutes, and the supernatant was collected (three times). 40 μL of 3M sodium acetate (pH 5.2) was added to the recovered solution, 440 μL of isopropanol was further added, and the mixture was quickly mixed and allowed to stand at −80 ° C. for 15 minutes. Next, the solution is centrifuged at 14,000 rpm for 4 minutes at 4 ° C., the supernatant is removed by pipetting, 400 μL of 70% ethanol is added, and further centrifuged at 14,000 rpm for 4 minutes at 4 ° C., and the supernatant is removed by pipetting. did. After desiccating the residual ethanol, 400 μL of RNase Free Water was added and gently mixed, and further 400 μL of 5M LiCl was added and gently mixed. After standing at 4 ° C. for 2 hours, centrifugation was performed at 4 ° C. and 12000 rpm for 15 minutes, and the supernatant was removed by pipetting. Furthermore, 400 μL of 70% ethanol was added again, and centrifugation was performed at 14,000 rpm at 4 ° C. for 5 minutes. After removing the supernatant by pipetting, the remaining ethanol was desiccated and 30 μL of RNase Free Water was added. The product was purified by RNA Mini elute to obtain the target genomic RNA.

１．２．インビトロ転写ゲノムＲＮＡ（インビトロＲＮＡ）の調製
鋳型ゲノムＲＮＡとして、Ａ２タンパク、コートタンパク、βサブユニットをコードする約４２１７塩基長のＲＮＡを用いた。ゲノムＲＮＡの調整はＳＫＱβプラスミドを用いたインビトロ転写により合成した。最初にＳＫＱβプラスミドをＨｉｎｄＩＩＩで３７℃１時間、制限酵素処理し、産物をＱｕｉａｇｅｎＱｕｉｃｋＣｏｌｕｍｎで精製し、アガロースゲル電気泳動（１％アガロースゲルＴＡＥｂｕｆｆｅｒ）で産物を確認した。次に、５’末端にＱβ ｇｅｎｏｍｅｐｒｉｍｅｒ＋３（配列番号６：TCTCTCTAATACGACTCACTATAGGGGGACCCCCTTTAGG）、３’末端にＱβ ｇｅｎｏｍｅｐｒｉｍｅｒ−３（配列番号７：CAAACCCGGGAGGAGAGAGGGCAAAGC）を結合させ、９４℃ ５分間後、９８度１０秒、６５℃３０秒、７２℃５分３０秒を３５サイクルでＰＣＲを行った。産物をＱｕｉａｇｅｎＱｕｉｃｋＣｏｌｕｍｎで精製し、アガロース電気泳動で確認後、ＳｍａＩで３７℃オーバーナイト制限酵素処理を行った。産物をＱｕｉａｇｅｎＱｕｉｃｋＣｏｌｕｍｎで精製し、ＲＮＡ合成を行った。反応条件は３７℃約５時間インキュベートし、その後、ＤＮａｓｅＩ（ＲＮａｓｅＦｒｅｅ）２ｕｌ加えさらに３７℃ １０〜２０分間インキュベートした。産物をＲＮＡＭｉｎｉｅｌｕｔｅで精製し目的のゲノムＲＮＡを得た。 1.2. Preparation of in vitro transcribed genomic RNA (in vitro RNA) As a template genomic RNA, RNA having a length of about 4217 bases encoding A2 protein, coat protein, and β subunit was used. Genomic RNA was synthesized by in vitro transcription using SKQβ plasmid. First, the SKQβ plasmid was treated with restriction enzyme with HindIII at 37 ° C. for 1 hour, the product was purified with Quiagen Quick Column, and the product was confirmed by agarose gel electrophoresis (1% agarose gel TAE buffer). Next, Qβ genome primer + 3 (SEQ ID NO: 6: TCTCTCTAATACGACTCACTATAGGGGGACCCCCTTTAGG) was bound to the 5 ′ end, and Qβ genome primer-3 (SEQ ID NO: 7: CAAACCCGGGAGGAGAGAGGGCAAAGC) was bound to the 3 ′ end. PCR was performed in 35 cycles of 30 ° C. and 72 ° C. for 5 minutes 30 seconds. The product was purified with Qiagen Quick Column, confirmed by agarose electrophoresis, and then treated with SmaI at 37 ° C. overnight restriction enzyme. The product was purified with Qiagen Quick Column and RNA synthesis was performed. The reaction conditions were incubation at 37 ° C. for about 5 hours, and then 2 ul of DNase I (RNase Free) was added and further incubated at 37 ° C. for 10 to 20 minutes. The product was purified by RNA Mini elute to obtain the target genomic RNA.

１．３．Ｓ２２２ＲＮＡの調製
鋳型ＲＮＡよりも短いＲＮＡとして、Ｓ２２２ＲＮＡを調製した。Ｓ２２２ＲＮＡは自然発生したＲＮＡをクローン化した２２２塩基長ＲＮＡである。Ｓ２２２ＲＮＡの調整にはｐＵＣ１９＿ｓ２２２Ａプラスミドを用いたインビトロ転写で合成した。最初にｐＵＣ１９ｋｈＦｐｒｉｍｅｒとｐＵＣ１９ｋｈＲ２ｐｒｉｍｅｒを用い、９４℃５分後、９８℃１０秒、５５℃３０秒、７２℃３０秒を３０サイクルでＰＣＲを行った。ＱｕｉａｇｅｎＱｕｉｃｋＣｏｌｕｍｎで精製後、アガロース電気泳動（１％アガロースゲルＴＡＥｂｕｆｆｅｒ）で産物を確認後、ＡｌｗＩで３７℃５時間制限酵素処理した。ＱｕｉａｇｅｎＱｕｉｃｋＣｏｌｕｍｎで精製し、産物をテンプレートとして、３７℃オーバーナイトでＲＮＡ合成を行った。その後、ＤＮａｓｅＩ（ＲＮａｓｅＦｒｅｅ）２ｕｌ加えさらに３７℃１０〜２０分間インキュベートした。産物をＲＮＡＭｉｎｉｅｌｕｔｅで精製し目的のＳ２２２ＲＮＡを得た。 1.3. Preparation of S222 RNA S222 RNA was prepared as RNA shorter than the template RNA. S222 RNA is a 222-base long RNA obtained by cloning a naturally occurring RNA. S222 RNA was prepared by in vitro transcription using pUC19_s222A plasmid. First, pUC19kh F primer and pUC19kh R2 primer were used, and after 94 ° C for 5 minutes, PCR was performed in 30 cycles of 98 ° C for 10 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds. After purification with Quiagen Quick Column, the product was confirmed by agarose electrophoresis (1% agarose gel TAE buffer), followed by restriction enzyme treatment with AlwI at 37 ° C. for 5 hours. Purification was performed with Qiagen Quick Column, and RNA synthesis was performed overnight at 37 ° C. using the product as a template. Thereafter, 2 ul of DNase I (RNase Free) was added and further incubated at 37 ° C. for 10 to 20 minutes. The product was purified with RNA Mini elute to obtain the desired S222 RNA.

２．油中水型エマルジョンの調製
２．１．油相成分の調製
エマルションの調整に用いた油相組成は、９５％ミネラルオイル（ＳＩＧＭＡＭ５９０４−５００ＭＬ）、４．５％ソルビタンモノオレエート（Ｗａｋｏ１９１−０８４８５）、０．５％ポリオキシエチレンソルビタンモノオレエート（Ｗａｋｏ１６１−２１６２１）。最初にソルビタンモノオレエートは粘度が高いので、誤差を小さくする目的で密度０．９８６ｇ／ｍＬを利用して質量から体積に換算し、その値を油相調整量の４．５％としてミネラルオイルを９５％相当加えＶＯＲＴＥＸ−ＧＥＮＩＥ２（ＳｃｉｅｎｔｉｆｉｃＩｎｄｕｓｔｒｉｅｓ）で約１分間混和した。次に０，５％相当のポリオキシエチレンソルビタンモノオレエートを加えＶＯＲＴＥＸ−ＧＥＮＩＥ２で約３０秒間混和した。反応液と同じ組成の飽和用バッファー（１２５ｍＭＴｒｉｓＣｌｐＨ７．８、５ｍＭＭｇＣｌ_２、０．０１％ＢＳＡ＆ＲＮａｓｅＦｒｅｅＷａｔｅｒ）を油相１ｍＬに対して１５０μＬ加え、ボルテックスで約１０秒間攪拌し、３７℃で約２０分間インキュベートした。インキュベート後１５０００ｒｐｍで約２０分間遠心分離を行い、油相を回収した。得られた油相を油中水型エマルション作製用の油相成分とした。 2. Preparation of water-in-oil emulsion 2.1. Preparation of oil phase component The oil phase composition used to prepare the emulsion was 95% mineral oil (SIGMA M5904-500ML), 4.5% sorbitan monooleate (Wako 191-08485), 0.5% polyoxyethylene sorbitan Monooleate (Wako 161-21621). First, sorbitan monooleate has a high viscosity, so in order to reduce the error, the density is converted from mass to volume using 0.986 g / mL, and the value is 4.5% of the oil phase adjustment amount. Was added in an amount equivalent to 95% and mixed with VORTEX-GENIE2 (Scientific Industries) for about 1 minute. Next, polyoxyethylene sorbitan monooleate corresponding to 0.5% was added, and the mixture was mixed with VORTEX-GENIE 2 for about 30 seconds. 150 μL of saturation buffer (125 mM TrisCl pH 7.8, 5 mM MgCl ₂ , 0.01% BSA & RNase Free Water) having the same composition as the reaction solution was added to 1 mL of the oil phase, and the mixture was vortexed for about 10 seconds and stirred at 37 ° C. Incubate for about 20 minutes. After the incubation, centrifugation was performed at 15000 rpm for about 20 minutes, and the oil phase was recovered. The obtained oil phase was used as an oil phase component for preparing a water-in-oil emulsion.

２．２．水相成分の調製
内封する反応液の組成は、１２５ｍＭＴｒｉｓＣｌｐＨ７．８、５ｍＭＭｇＣｌ_２、０．０１％ＢＳＡ、１．２５ｍＭ各ＮＴＰｓである。希釈は全てＲＮａｓｅＦｒｅｅＷａｔｅｒで行った。さらにゲノムＲＮＡやＱβレプリカーゼ（β−サブユニット、ＥＦ−ＴＵ、ＥＦ−ＴＳ）、Ｓ１タンパク、Ｈｆｑタンパクはそれぞれの実験に応じて加えた。 2.2. Preparation of aqueous phase component The composition of the reaction solution to be enclosed is 125 mM TrisCl pH 7.8, 5 mM MgCl ₂ , 0.01% BSA, 1.25 mM NTPs. All dilutions were done with RNase Free Water. Furthermore, genomic RNA, Qβ replicase (β-subunit, EF-TU, EF-TS), S1 protein, and Hfq protein were added according to each experiment.

２．３．油中水型エマルジョンの調製
油中水型エマルションの作製は１．５ｍＬエッペンチューブで行った。上記で調整した油相成分４８０μＬに対して、水相成分２０μＬを添加し、ＶＯＲＴＥＸ−ＧＥＮＩＥ２で約３０秒間攪拌した。攪拌の際、目視で均一に混ざっていることを確認した。また、攪拌強度は機械目盛り８で統一した。また、メンブレンを使った油中水型エマルションの作製も行った。上記で調整した油相９６０μＬに対して、反応液（水相）４０μＬ加え、攪拌は２０往復で統一し作製した。油中水型エマルションの作製は全て４℃条件下で行った。 2.3. Preparation of water-in-oil emulsion A water-in-oil emulsion was prepared in a 1.5 mL Eppendorf tube. 20 μL of aqueous phase component was added to 480 μL of the oil phase component adjusted as described above, and the mixture was stirred for about 30 seconds with VORTEX-GENIE2. Upon stirring, it was visually confirmed that they were uniformly mixed. Further, the stirring intensity was unified on the mechanical scale 8. In addition, a water-in-oil emulsion using a membrane was also prepared. 40 μL of the reaction liquid (aqueous phase) was added to 960 μL of the oil phase adjusted as described above, and stirring was performed in 20 reciprocations. All the water-in-oil emulsions were prepared under 4 ° C conditions.

２．４．油中水型エマルジョンからの水相成分の回収
最初にエマルション溶液に複製反応を停止させる目的で１００ｍＭＥＤＴＡ（Ｗａｋｏ）を２μｌ加え、その上からジエチルエーテル（Ｗａｋｏ）を油相の１／２量加え混和した。この際、１／２量より多くジエチルエーテルを加えると、界面活性剤が析出してしまい水相の回収が困難になるので注意が必要である。混和後、１７０００ｒｐｍ、４℃で５分間遠心分離操作を行い、上層のエーテルと油相の混和物を除去した。さらに油相と等量のジエチルエーテルを加え混和し、１００００ｒｐｍ、４℃で２分間遠心分離操作後、上層のエーテルと残存油相の混和物を除去した。この際、回収産物の濃度を正確に定量するためにエーテルは極力取り除くことが望ましい。さらに残存エーテルを完全に除去するため、氷上で約１５分間揮発させ、下層の水相を回収した。 2.4. Recovery of water phase component from water-in-oil emulsion First, 2 μl of 100 mM EDTA (Wako) was added to the emulsion solution for the purpose of stopping the replication reaction, and then diethyl ether (Wako) was added in half the amount of the oil phase. Mixed. At this time, if more diethyl ether is added than 1/2 amount, the surfactant is precipitated and it is difficult to recover the aqueous phase. After mixing, the mixture was centrifuged at 17000 rpm and 4 ° C. for 5 minutes to remove the mixture of the upper ether layer and the oil phase. Further, the same amount of diethyl ether as that of the oil phase was added and mixed. After centrifugation at 10,000 rpm and 4 ° C. for 2 minutes, the mixture of the upper ether layer and the remaining oil phase was removed. At this time, it is desirable to remove ether as much as possible in order to accurately determine the concentration of the recovered product. Further, in order to completely remove the remaining ether, it was volatilized on ice for about 15 minutes, and the lower aqueous phase was recovered.

３．反応産物の測定
電気泳動、ＳＤＳ−ＰＡＧＥ写真からの酵素、ＲＮＡ濃度計算は、画像解析ソフトＩｍａｇｅＪで行った。まず編集項目で画像データの色を反転させ、測定範囲をなるべくバンドの大きさに合わせ、強度を測定した。その後ブランクとして同じ測定範囲でバンドと同レーンのゲル部分を測定した。データとしては、面積と平均強度の数値が得られるので、その積を算出、ブランクを差し引いた値を定量用の数値とした。 3. Measurement of reaction products Electrophoresis, enzyme from SDS-PAGE photographs, and RNA concentration calculation were performed with image analysis software Image J. First, the color of the image data was reversed in the edit item, the measurement range was matched to the size of the band as much as possible, and the intensity was measured. Thereafter, the gel portion of the band and the same lane was measured in the same measurement range as a blank. As data, numerical values of area and average intensity were obtained, so the product was calculated and the value obtained by subtracting the blank was used as the numerical value for quantification.

４．ｑＲＴ−ＰＣＲ
エマルジョンから回収した反応産物内のゲノムＲＮＡ＋鎖、−鎖の定量を行った。逆転写反応が阻害されないＳＤＳ量が０．０００５％以下ということが報告されているので、それに準拠して試薬の調整を行った。まず２本鎖をほどく目的で、サンプルを１ｍＭＥＤＴＡで１０００倍希釈しＳＤＳを０．０００５％になるように加え、９５℃ ５分間熱処理を行った。その後、１サンプルを２本に分注し、それぞれ＋鎖検出用プライマー（１ｓｔＲＴａｎｎｅａｌＲｐｒｉｍｅｒ）と−鎖検出用プライマー（１ｓｔＲＴａｎｎｅａｌＦｐｒｉｍｅｒ）を結合させ、５０℃ ３０分間、７０℃ １５分間で逆転写した。反応後のサンプルを１００倍希釈し、＋鎖検出には２ｎｄＴＰＣＲｐｒｉｍｅｒとＭＢＴＦｐｒｉｍｅｒ、−鎖検出には２ｎｄＴＰＣＲｐｒｉｍｅｒとＭＢＴＲｐｒｉｍｅｒを使ってＴａｑｍａｎｐｒｏｖｅによる定量ＰＣＲをＭｘ３００５Ｐ（ＳＴＲＡＴＡＧＥＮＥ）で行った（９５℃ １０分後、９５℃ １５秒／６０℃ １分／５０サイクル）。 4). qRT-PCR
Quantification of the genomic RNA + strand and − strand in the reaction product recovered from the emulsion was performed. Since it has been reported that the amount of SDS that does not inhibit the reverse transcription reaction is 0.0005% or less, the reagent was adjusted in accordance with it. First, in order to unwind the double strand, the sample was diluted 1000 times with 1 mM EDTA, SDS was added to 0.0005%, and heat treatment was performed at 95 ° C. for 5 minutes. Thereafter, one sample is dispensed into two, and a + strand detection primer (1st RT annealing R primer) and a − strand detection primer (1st RT annealing F primer) are combined, and 50 ° C. for 30 minutes, 70 ° C. 15 Reverse transcription in minutes. The sample after the reaction was diluted 100-fold, and quantitative PCR by Taqman probe was performed with Mx 3005P (STRATA GENE) using 2nd TPCR primer and MBTF primer for + strand detection, and 2nd TPCR primer and MBTR primer for -strand detection. (95 ° C 10 minutes later, 95 ° C 15 seconds / 60 ° C 1 minute / 50 cycles).

５．２本鎖率の測定
ゲノムＲＮＡ複製反応を行い、電気泳動で産物を確認した際、目的産物ｓｓＲＮＡの上方に常にもう１本バンドが現れる。これは２本鎖だといわれているが、過去の論文に従ってそのことを確認した。まずサンプルをＴＥバッファーで９５℃ ５分間熱処理し、アニーリングバッファーを加え、６５℃で１時間アニーリングを行った。サンプル４ｕｌに対して、ＲＮａｓｅｆｒｅｅＷａｔｅｒ２ｕｌ、１０ｘＬｏａｄｉｎｇｂｕｆｆｅｒ（ＴａＫａＲａ）１ｕｌ加え希釈し、６ｕｌを１％ＴＡＥアガロースゲル（Ａｇａｒｏｓｅ−ＨＧＳ：ｎａｋａｌａｉｔｅｓｑｕｅ）、泳動ｂｕｆｆｅｒＴＡＥで１００Ｖ約３０分間電気泳動した。電気泳動後、１ｘＳＹＢＥＲ−ＧｒｅｅｎＩＩＲＮＡＧｅｌＳｔａｉｎｓ（ＴａＫａＲａ）で約３０分間振とうさせながら染色後、Ｔｙｐｈｏｏｎ９２１０（ＡｍｅｒｓｈａｍＢｉｏｓｃｉｅｎｃｅｓ）で蛍光を測定した。バンド強度を画像解析ソフトＩｍａｇｅＪで測定し、その値からｄｓＲＮＡ／（ｓｓＲＮＡ＋ｄｓＲＮＡ）を算出し、２本鎖率を測定した。 5. Measurement of double-strand rate When a genomic RNA replication reaction is performed and the product is confirmed by electrophoresis, another band always appears above the target product ssRNA. This is said to be double-stranded, but I confirmed it according to past papers. First, the sample was heat-treated with TE buffer at 95 ° C. for 5 minutes, an annealing buffer was added, and annealing was performed at 65 ° C. for 1 hour. 4 ul of sample was diluted by adding 1 ul of RNase free water 2 ul, 10 × loading buffer (TaKaRa), and 6 ul was electrophoresed with 1% TAE agarose gel (Agarose-HGS: nakarai tesque) and electrophoresis buffer TAE for about 30 minutes. After electrophoresis, staining was performed while shaking with 1 × SYBER-GreenII RNA Gel Stains (TaKaRa) for about 30 minutes, and then fluorescence was measured with Typhoon 9210 (Amersham Biosciences). The band intensity was measured with image analysis software Image J, dsRNA / (ssRNA + dsRNA) was calculated from the value, and the double-stranded rate was measured.

（実施例１：油中水型エマルジョン内でのゲノムＲＮＡの複製）
（１）油中水型エマルジョン調製時の飽和バッファーの必要性
油中水型エマルジョン内で反応を行う際、内封反応液（水相）の外相（油相）へのもれ込みが生じ、内部反応が阻害されることが予想されたので、これを防ぐために内封反応液と同じ組成のバッファーで油相を飽和させるといった操作を行うことにした。結果、飽和操作ありだと、目的産物であるssRNAの増幅が確認できたが、飽和操作なしでは全体的にバンド強度が弱く反応が進んでいないことがわかった。 (Example 1: Replication of genomic RNA in water-in-oil emulsion)
(1) Necessity of a saturated buffer when preparing a water-in-oil emulsion When reaction is carried out in a water-in-oil emulsion, leakage of the encapsulated reaction liquid (water phase) into the outer phase (oil phase) occurs. Since the internal reaction was expected to be inhibited, in order to prevent this, it was decided to perform an operation such as saturating the oil phase with a buffer having the same composition as the enclosed reaction solution. As a result, when the saturation operation was performed, amplification of the target product ssRNA could be confirmed, but it was found that the band intensity was weak overall and the reaction did not proceed without the saturation operation.

（２）メンブレンとボルテックスで作製した油中水型エマルションのサイズ
メンブレンとボルテックスで油中水型エマルションを作製することで、そのサイズがどのよう異なるか確認した。油中水型エマルションの油相、水相組成、及び調整法は上記と同じように作製した。それぞれのエマルション溶液を顕微鏡で観察し、ランダムで画像を10枚撮影、それを画像解析ソフトNI visionで個数を数え、サイズ分布を算出した。結果、メンブレンは3種類とも分散が小さく、均一な油中水型エマルションができていることがわかった。またポアサイズ20μmでは直径約1μm、ポアサイズ15μmでは直径約1.5μm、ポアサイズ5μmでは直径約2μmの油中水型エマルションが大部分を占めていることがわかった。ボルテックスを用いた場合、分散が大きく、直径が約10μmの油中水型エマルションが大部分を占めていることがわかった。 (2) Size of water-in-oil emulsion prepared with membrane and vortex By making a water-in-oil emulsion with membrane and vortex, it was confirmed how the sizes differ. The oil phase, water phase composition, and adjustment method of the water-in-oil emulsion were prepared in the same manner as described above. Each emulsion solution was observed with a microscope, 10 images were taken at random, the number was counted with the image analysis software NI vision, and the size distribution was calculated. As a result, it was found that all three types of membranes had a small dispersion and a uniform water-in-oil emulsion was formed. It was also found that water-in-oil emulsions with a pore size of 20 μm accounted for approximately 1 μm in diameter, pore sizes of 15 μm with a diameter of approximately 1.5 μm, and pore sizes of 5 μm with a diameter of approximately 2 μm. When vortexing was used, it was found that water-in-oil emulsions with large dispersion and a diameter of about 10 μm accounted for the majority.

（３）メンブレンとボルテックスの比較
メンブレン、ボルテックスで作製した油中水型エマルション内でQβレプリカーゼ１によるゲノムRNA複製反応が進行するかどうかを確認した。3回行った結果、ゲノムRNA複製量はボルテックス＞ポアサイズ20μmのメンブレン＞ポアサイズ10μmのメンブレン＞ポアサイズ5μmのメンブレン＞試験管内の順であった。またspRNAの複製量は、ポアサイズ20μmのメンブレン＜ポアサイズ10μmのメンブレン≒ポアサイズ5μmのメンブレン＜ボルテックス＜試験管内の順であった。メンブレンを使った場合、ポアサイズ10μmのメンブレン、ポアサイズ5μmのメンブレンを用いた場合、RNAの分解と思われるバンドのスメアが見られた。また油中水型エマルションのサイズが小さいほどspRNAが抑えられると予想していたが、それに反してポアサイズ10μmのメンブレンおよびポアサイズ5μmのメンブレンにおいてspRNAの増加が確認された。 (3) Comparison between membrane and vortex It was confirmed whether or not the genomic RNA replication reaction by Qβ replicase 1 proceeded in a water-in-oil emulsion prepared by membrane and vortex. As a result of performing three times, the amount of genomic RNA replication was in the order of vortex> membrane with a pore size of 20 μm> membrane with a pore size of 10 μm> membrane with a pore size of 5 μm> in a test tube. The amount of spRNA replicated was in the order of a membrane with a pore size of 20 μm <a membrane with a pore size of 10 μm≈a membrane with a pore size of 5 μm <vortex <in a test tube. When membranes were used, smears of bands that were considered to be RNA degradation were observed when membranes with a pore size of 10 μm and membranes with a pore size of 5 μm were used. In addition, it was expected that spRNA was suppressed as the size of the water-in-oil emulsion was smaller. On the contrary, an increase in spRNA was confirmed in the membrane having a pore size of 10 μm and the membrane having a pore size of 5 μm.

（４）S222 RNAを使ったW/Oエマルション内RNA複製反応の経時変化
過去の研究においてRNA複製反応の速度論解析は試験管内反応の結果から算出されてきた。しかし、本研究では反応場はW/Oエマルション内であり、試験管内とは異なる環境であるため、そのことによる酵素反応への影響を確認する必要がある。そこで、W/Oエマルション内でも試験管内反応と同様にRNA複製反応が起こるかどうかをS222 RNAを使って確認した。S222 RNAはspRNAを単離しクローン化したRNAであるため、複製反応過程でのspRNAによる反応阻害は起こらない。試験管内でのQβレプリカーゼ１によるS222 RNA (spRNA)複製量とW/Oエマルション内でのRNAの複製量を比較することで、W/Oエマルション内と試験管内とで複製反応の進行が異なるのかどうかを確認した。メンブレン、ヴォルテックス双方で作製したW/OエマルションでS222 RNA複製反応を行い、それぞれ0分、5分、10分、20分、30分のRNA複製量を比較した。その結果、試験管内反応とヴォルテックスで作製したW/Oエマルション内反応ではRNA複製量に差が見られなかった。このことからヴォルテックスで作製したW/Oエマルションは酵素に影響せず、試験管反応時と同等に進行していることがわかった。逆にメンブレンで作製したW/Oエマルション内反応ではRNA複製量が他2つにくらべて約1/2になった。メンブレンで作製したW/Oエマルションでは酵素反応が十分に進行しないことが示された。 (4) Time course of RNA replication reaction in W / O emulsion using S222 RNA In previous studies, kinetic analysis of RNA replication reaction has been calculated from the results of in vitro reaction. However, in this study, the reaction field is in the W / O emulsion, and the environment is different from that in the test tube, so it is necessary to confirm the effect on the enzyme reaction. Therefore, S222 RNA was used to confirm whether RNA replication reaction occurred in W / O emulsion as well as in vitro reaction. Since S222 RNA is RNA obtained by isolating and cloning spRNA, reaction inhibition by spRNA does not occur during the replication reaction. By comparing the amount of S222 RNA (spRNA) replicated by Qβ replicase 1 in vitro and the amount of RNA replicated in W / O emulsion, does the replication reaction progress differently in W / O emulsion and in test tube? I confirmed. S222 RNA replication reaction was performed using W / O emulsions prepared by both membrane and vortex, and RNA replication amounts were compared at 0 min, 5 min, 10 min, 20 min, and 30 min, respectively. As a result, there was no difference in RNA replication between the reaction in vitro and the reaction in W / O emulsion prepared by Vortex. From this, it was found that the W / O emulsion prepared by Vortex did not affect the enzyme and progressed as much as during the test tube reaction. Conversely, in the W / O emulsion reaction prepared with the membrane, the RNA replication amount was about ½ compared to the other two. It was shown that the enzyme reaction did not proceed sufficiently in the W / O emulsion prepared with the membrane.

結果を図１に示す。図１は、Ｓ２２２ＲＮＡを、試験管内（◆）、ボルテックスを用いて調製した油中水型エマルジョン（「Ｗ／Ｏエマルジョン」ともいう）内（○）、および、メンブレンを用いて調製したＷ／Ｏエマルジョン（●）を用いて増幅した結果を示す。実験条件は、以下のとおりである：所期Ｓ２２２ＲＮＡ濃度１０ｎＭ；Ｑβレプリカーゼ１濃度１００ｎＭ；Ｓ１タンパク２００ｎＭ；Ｈｆｑタンパク６００ｎＭ；油相量メンブレンを用いて調製したＷ／Ｏエマルジョンの場合９６０μＬ、ヴォルテックスを用いて調製したＷ／Ｏエマルジョンの場合４８０μＬ；水相量メンブレンを用いて調製したＷ／Ｏエマルジョンの場合４０μＬ、ヴォルテックスを用いて調製したＷ／Ｏエマルジョンの場合２０μＬ；検出系電気泳動（１０％ポリアクリルアミドゲル）；反応条件３７℃ ３０分間。 The results are shown in FIG. FIG. 1 shows S222 RNA in a test tube (♦), a water-in-oil emulsion prepared using a vortex (also referred to as “W / O emulsion”) (◯), and a W / W prepared using a membrane. The results of amplification using O emulsion (●) are shown. The experimental conditions were as follows: Expected S222 RNA concentration 10 nM; Qβ replicase 1 concentration 100 nM; S1 protein 200 nM; Hfq protein 600 nM; oil phase amount 480 μL in the case of W / O emulsion prepared using 40 μL in the case of W / O emulsion prepared using membrane, 20 μL in case of W / O emulsion prepared using vortex; detection system electrophoresis (10% Polyacrylamide gel); reaction conditions 37 ° C. for 30 minutes.

（５）メンブレン使用時にＲＮＡ複製量が低くなる理由の解明
メンブレンでＷ／Ｏエマルションを作製し、その内部でＲＮＡ複製反応を行うと反応速度と複製量が低下するといった結果が得られた。Ｗ／Ｏエマルション内ゲノムＲＮＡ複製反応の速度論解析を行う上で試験管内反応と進行具合が異なることは過去の研究結果等と比較する上で問題であり、実験の信憑性に関わる問題となり得る。よってその原因の解明と改善を試みた。まずその低下の原因はｉ）酵素の失活、ｉｉ）実験操作による有効酵素濃度の低下の２仮説が考えられた。この２項目を確認するために、反応後の酵素量をＳＤＳ−ＰＡＧＥで確認した。酵素量に差が見られなければ仮説ｉ）が原因であり、差があれば仮説ｉｉ）が原因であると考えた。 (5) Elucidation of the reason that the amount of RNA replication is reduced when the membrane is used When a W / O emulsion was prepared with a membrane and RNA replication reaction was carried out inside the membrane, the reaction rate and the amount of replication were reduced. The difference in progress from in vitro reaction in the kinetic analysis of genomic RNA replication reaction in W / O emulsion is a problem in comparison with past research results, etc., and may be a problem related to the authenticity of the experiment . Therefore, we tried to elucidate and improve the cause. First, two hypotheses were considered: i) inactivation of the enzyme and ii) reduction of the effective enzyme concentration due to the experimental operation. In order to confirm these two items, the amount of enzyme after the reaction was confirmed by SDS-PAGE. If there was no difference in the amount of enzyme, hypothesis i) was the cause, and if there was a difference, hypothesis ii) was the cause.

結果、メンブレンで作製したW/Oエマルション内では酵素濃度が低下していた。このことからｉｉ）実験操作による有効酵素濃度の低下が示唆された。それはメンブレン孔に酵素が凝集し詰まっていることが原因だと考えられたので、このことを確認するために、（ａ）反応液中にＤＴＴを加えることにより、酵素の凝集が回避され、メンブレン孔に詰まらなくなるか否かを、検討した。また、（ｂ）従来の材質が親水性であることから、疎水性メンブレンを用いることによりレプリカーゼの吸着量が減少するか否かについても、検討した。Ｗ／Ｏエマルション内でＳ２２２ＲＮＡ複製反応を行った場合の複製量もまた、検討した。 As a result, the enzyme concentration was reduced in the W / O emulsion prepared with the membrane. From this, it was suggested that ii) the effective enzyme concentration was lowered by the experimental procedure. It was thought that this was due to the fact that the enzyme was agglomerated and clogged in the membrane pores. In order to confirm this, (a) by adding DTT to the reaction solution, the aggregation of the enzyme was avoided, and the membrane It was examined whether or not the holes could be clogged. In addition, (b) since the conventional material is hydrophilic, whether or not the adsorption amount of replicase is reduced by using a hydrophobic membrane was also examined. The amount of replication when the S222 RNA replication reaction was performed in a W / O emulsion was also examined.

結果、ＤＴＴを加えることで、メンブレン使用時のβサブユニット回収量が試験管、ヴォルテックスの場合で、ほぼ同等となった。メンブレンを疎水性メンブレンに交換しても、βサブユニット回収量は増えなかった。結果を図２に示す。ＲＮＡ複製結果を図３に示す。 As a result, by adding DTT, the β subunit recovery amount when using the membrane was almost the same in the case of the test tube and the vortex. Replacing the membrane with a hydrophobic membrane did not increase the amount of β subunit recovered. The results are shown in FIG. The RNA replication results are shown in FIG.

ＤＴＴを加えることでβサブユニット回収量は大幅に上がったが、メンブレンを用いた場合は、ＲＮＡ複製量は試験管、ヴォルテックスと比較して、これらの約１／２程度だった。また疎水性メンブレンではＲＮＡ複製量の増加は見られなかった（図２）。以上をふまえて、酵素のメンブレン孔への吸着、凝集以外にもなんらかのメンブレン特有の反応阻害があることが示唆された。原因の解明はできなかったが、ヴォルテックスで作製したＷ／Ｏエマルション内ＲＮＡ複製反応は試験管内反応と同等の結果を示したことから、以後ヴォルテックスでＷ／Ｏエマルションを作製し実験を進めることにした。 Although the amount of β subunit recovered significantly increased by adding DTT, the amount of RNA replication was about ½ of these when compared to test tubes and vortexes when membranes were used. In addition, no increase in the amount of RNA replication was observed in the hydrophobic membrane (FIG. 2). Based on the above, it was suggested that there was some kind of membrane-specific reaction inhibition other than the adsorption and aggregation of the enzyme into the membrane pores. Although the cause could not be clarified, the RNA replication reaction in W / O emulsion prepared by Vortex showed the same result as the reaction in vitro. did.

（６）ゲノムＲＮＡの決定
Ｑβファージから抽出したＲＮＡ（インビボＲＮＡ）には鋳型となるＲＮＡの他に、もう２種類のＲＮＡが存在することが電気泳動により確認されている。この目的外のＲＮＡが反応に何かしら影響を及ぼすのではないか、また反応産物の正確な定量ができないことが予想されたので、この２種類のＲＮＡが存在しないＲＮＡ（インビトロＲＮＡ）を作製し、このＲＮＡで複製反応が起こるかどうかを確認した。その結果、インビトロＲＮＡを用いた場合には、ＲＮＡが問題なく増幅したので、以後このインビトロＲＮＡを使いた。 (6) Determination of genomic RNA It has been confirmed by electrophoresis that RNA extracted from Qβ phage (in vivo RNA) contains two types of RNA in addition to the template RNA. Since it was expected that the RNA other than this purpose would have some influence on the reaction, and it was expected that the reaction product could not be accurately quantified, an RNA without these two types of RNA (in vitro RNA) was prepared, It was confirmed whether a replication reaction occurred with this RNA. As a result, when in vitro RNA was used, RNA was amplified without any problem, and this in vitro RNA was used thereafter.

（７）Ｑβレプリカーゼの決定
Ｑβレプリカーゼ１とＱβレプリカーゼ２とを、ＲＮＡ増幅能について比較した。結果、ｓｓＲＮＡの増幅量は双方変わらなかったが、Ｑβレプリカーゼ１ではＱβレプリカーゼ２に比べてｓｐＲＮＡが多く出たので、以後の実験ではＱβレプリカーゼ２を使いた。 (7) Determination of Qβ replicase Qβ replicase 1 and Qβ replicase 2 were compared for RNA amplification ability. As a result, the amount of amplification of ssRNA did not change, but since Qβ replicase 1 produced more spRNA than Qβ replicase 2, Qβ replicase 2 was used in subsequent experiments.

（８）反応液中の希釈緩衝液量比の決定
希釈緩衝液はＱβレプリカーゼやＳ１タンパク、Ｈｆｑタンパクを保存するためのバッファーでグリセロールが含まれている。ＲＮＡ複製反応においてグリセロールは反応を阻害することが知られているので極力少なくしたほうがよいとされており、過去の報告によれば、全体量の１／２０が理想とされている。しかし、ピペッティング誤差を小さくするためには反応液２０μＬに対して２μＬが適当だと考えられたので、全体量の１／１０にすることによる反応阻害の度合いを確認した。また同時に、あらかじめ活性の低い酵素を完全に失活させるプレインキュベーション操作（反応液の調整時に３７℃３０分間インキュベート）を行えば、より正確なＲＮＡ複製反応の様子を確認できると考えられたため、プレインキュベーション操作のありなしでＲＮＡ複製反応にどう影響するかを確認した。結果、希釈緩衝液量を２μＬにすることによる反応への悪影響はなく、従来どおりプレインキュベーションも行うことにした。 (8) Determination of dilution buffer volume ratio in reaction solution The dilution buffer is a buffer for storing Qβ replicase, S1 protein, and Hfq protein, and contains glycerol. Since it is known that glycerol inhibits the reaction in RNA replication reaction, it is recommended to reduce it as much as possible. According to past reports, 1/20 of the total amount is ideal. However, in order to reduce the pipetting error, 2 μL was considered appropriate for 20 μL of the reaction solution, so the degree of reaction inhibition by reducing the total amount to 1/10 was confirmed. At the same time, it was considered that a more accurate RNA replication reaction could be confirmed by performing a preincubation operation (incubation at 37 ° C. for 30 minutes when preparing the reaction solution) to completely inactivate the low activity enzyme in advance. It was confirmed how the RNA replication reaction was affected with and without incubation. As a result, there was no adverse effect on the reaction by setting the dilution buffer volume to 2 μL, and preincubation was also performed as usual.

（９）Ｓ１タンパク最適濃度の決定
Ｑβレプリカーゼは、前述したようにβサブユニットとＥＦ−ＴＵ、ＥＦ−ＴＳの３量体に外部因子のＳ１タンパクが作用して４量体で働く。そこで、よりゲノムＲＮＡが増幅されるＳ１タンパクの最適濃度を決定した。３量体濃度を２４０ｎＭ、Ｈｆｑ濃度を６００ｎＭに固定し、Ｓ１濃度を０、２５、５０、１００、２００ｎＭとしてＷ／Ｏエマルションを調製し、各々について、ＲＮＡ複製反応を測定した。３回行った結果、Ｓ１タンパク濃度に依存してゲノムＲＮＡ複製量が増加することが示された。よってＱβレプリカーゼに対して十分量にあたる２００ｎＭを最適濃度とした。 (9) Determination of optimum S1 protein concentration As described above, Qβ replicase works as a tetramer with the S1 protein of the external factor acting on the β subunit, EF-TU, and EF-TS trimer. Therefore, the optimum concentration of S1 protein at which genomic RNA is amplified is determined. W / O emulsions were prepared with the trimer concentration fixed at 240 nM, the Hfq concentration fixed at 600 nM, and the S1 concentrations at 0, 25, 50, 100, and 200 nM, and the RNA replication reaction was measured for each. As a result of performing three times, it was shown that the amount of genomic RNA replication increased depending on the S1 protein concentration. Therefore, 200 nM, which is a sufficient amount with respect to Qβ replicase, was determined as the optimum concentration.

（１０）Ｈｆｑタンパク最適濃度の決定
Ｈｆｑタンパクは６量体タンパクでゲノムＲＮＡ複製反応を促進する働きがあることは知られているが、その詳細はまだ良くわかっていない。そこで、よりゲノムＲＮＡが増幅されるＨｆｑタンパクの最適濃度を決定した。３量体濃度を２４０ｎＭ、Ｓ１濃度を２００ｎＭに固定し、Ｈｆｑ濃度を０、６０、１５０、３００、６００、１２００ｎＭとして、各々についてＷ／Ｏエマルションを調製し、ＲＮＡ複製反応を測定した。３回行った結果、Ｈｆｑタンパクは少なくても、多すぎてもゲノムＲＮＡが増えにくくなることが示された。また３００〜６００ｎＭが最適濃度であることがわかった。よって３００ｎＭを本実験での最適濃度とした。結果を図４に示す。反応条件は、以下のとおりである：所期インビトロＲＮＡ濃度１０ｎＭ；Ｑβレプリカーゼ２濃度２４ｎＭ；Ｓ１タンパク質濃度２００ｎＭ；エマルジョンの作製ＶＯＲＴＥＸ−ＧＥＮＩＥ２を用いた；反応時間３７℃ ４０分間；検出系電気泳動（１％ＴＥＡアガロース）。 (10) Determination of optimum concentration of Hfq protein It is known that Hfq protein is a hexameric protein and has a function of promoting a genomic RNA replication reaction, but details thereof are not well understood. Therefore, the optimum concentration of Hfq protein at which genomic RNA was amplified was determined. W / O emulsions were prepared for each of the trimer concentration at 240 nM, the S1 concentration at 200 nM, and the Hfq concentrations at 0, 60, 150, 300, 600, and 1200 nM, and RNA replication reaction was measured. As a result of performing three times, it was shown that the amount of genomic RNA hardly increases even if the amount of Hfq protein is small or too large. It was also found that 300 to 600 nM was the optimum concentration. Therefore, 300 nM was set as the optimum concentration in this experiment. The results are shown in FIG. Reaction conditions are as follows: Initial in vitro RNA concentration 10 nM; Qβ replicase 2 concentration 24 nM; S1 protein concentration 200 nM; Emulsion preparation VORTEX-GENIE2 was used; Reaction time 37 ° C. 40 minutes; Detection system electrophoresis ( 1% TEA agarose).

（実施例２：Ｗ／Ｏエマルション内でＱβレプリカーゼによるゲノムＲＮＡ複製反応の速度論解析）
（１）指数期の解析
酵素がＲＮＡの対して過剰に存在する場合、全てのＲＮＡに酵素が結合していると仮定されるため、ＲＮＡは指数的に増幅するはずである。そこでゲノムＲＮＡ（インビトロＲＮＡ）２ｎＭに対してＱβレプリカーゼ２２４０ｎＭ（ただし、約１０％が活性を保持し、約９０％は失活していた）の条件化で、Ｗ／Ｏエマルション内で反応させ、その経時変化を観察した。サンプリングタイムは０、５、１０、１５、２０、３０、４０、５０分の点で行った。結果を図５に示す。図５Ｂは、図５Ａの縦軸を、指数表記したものである。反応条件は、以下のとおりである：所期インビトロＲＮＡ濃度１０ｎＭ；Ｑβレプリカーゼ２濃度２４ｎＭ；Ｓ１タンパク質２００ｎＭ；Ｈｆｑタンパク質３００ｎＭ；エマルジョンの作製ＶＯＲＴＥＸ−ＧＥＮＩＥ２を用いた；検出系電気泳動（１％ＴＥＡアガロース）。 (Example 2: Kinetic analysis of genomic RNA replication reaction by Qβ replicase in W / O emulsion)
(1) Analysis of the exponential phase If the enzyme is present in excess relative to the RNA, it should be amplified exponentially since it is assumed that the enzyme is bound to all RNA. Therefore, the reaction was performed in a W / O emulsion under conditions of Qβ replicase 2 240 nM (however, about 10% retained activity and about 90% was inactivated) against 2 nM genomic RNA (in vitro RNA). The change with time was observed. The sampling time was 0, 5, 10, 15, 20, 30, 40, and 50 minutes. The results are shown in FIG. FIG. 5B is an exponential representation of the vertical axis of FIG. 5A. Reaction conditions are as follows: Initial in vitro RNA concentration 10 nM; Qβ replicase 2 concentration 24 nM; S1 protein 200 nM; Hfq protein 300 nM; Preparation of emulsion Using VORTEX-GENIE2; Detection system Electrophoresis (1% TEA agarose ).

この結果について下記のモデル The following model for this result

に従いｋｃａｔを算出すると、ｋｃａｔ＝０．２４±０．０３であった。 Kcat was calculated according to the following equation, kcat = 0.24 ± 0.03.

（２）線形期の解析
ＲＮＡが酵素に対して過剰に存在する場合、全ての酵素にＲＮＡが結合していると仮定されるため、ＲＮＡは線形的に増幅する。そこでゲノムＲＮＡ（インビトロＲＮＡ）５０ｎＭに対してＱβレプリカーゼ２２４０ｎＭ（ただし、約１０％が活性を保持し、約９０％は失活していた）の条件化で、Ｗ／Ｏエマルション内で反応させ、その経時変化を観察した。サンプリングタイムは０、５、１０、１５、２０、３０、４０、５０分の点で行った。結果を図６に示す。反応条件は、以下のとおりである：所期インビトロＲＮＡ濃度１０ｎＭ；Ｑβレプリカーゼ２濃度２４ｎＭ；Ｓ１タンパク質２００ｎＭ；Ｈｆｑタンパク質３００ｎＭ；エマルジョンの作製ＶＯＲＴＥＸ−ＧＥＮＩＥ２を用いた；検出系電気泳動（１％ＴＥＡアガロース）。 (2) Analysis of the linear phase When RNA is present in excess relative to the enzyme, it is assumed that RNA is bound to all enzymes, and thus RNA is linearly amplified. Therefore, the reaction was performed in a W / O emulsion under the condition of Qβ replicase 2 240 nM (however, about 10% retained activity and about 90% deactivated) against 50 nM genomic RNA (in vitro RNA). The change with time was observed. The sampling time was 0, 5, 10, 15, 20, 30, 40, and 50 minutes. The results are shown in FIG. Reaction conditions are as follows: Initial in vitro RNA concentration 10 nM; Qβ replicase 2 concentration 24 nM; S1 protein 200 nM; Hfq protein 300 nM; Preparation of emulsion Using VORTEX-GENIE2; Detection system Electrophoresis (1% TEA agarose ).

この結果について下記のモデル The following model for this result

に従いｋｃａｔを算出すると、ｋｃａｔ＝０．１０３±０．０１２であった。 Kcat was calculated according to the equation, kcat = 0.103 ± 0.012.

（３）ｑＲＴ−ＰＣＲによる＋鎖、−鎖の定量
Ｗ／Ｏエマルション内ゲノムＲＮＡ複製反応で回収したサンプルについて、定量ＲＴ−ＰＣＲを用いて、＋鎖、−鎖の定量を行った。全てのサンプルは電気泳動で定量した濃度から５ｎＭになるように希釈して使った。スタンダード濃度を５０、５、０．５ｎＭとした。結果、＋鎖、−鎖の合計は電気泳動からの定量結果と大きくずれたが、全体的な傾向としては、＋鎖より−鎖の方が少ないといった結果が得られた。指数期サンプルを用いた結果を図７Ａに、線形期サンプルを用いた結果を図７Ｂに示す。電気泳動での測定値を（■）、ｑＲＴ−ＰＣＲの＋鎖と−鎖の合計を（□）、ｑＲＴ−ＰＣＲの＋鎖を（●）、ｑＲＴ−ＰＣＲの−鎖を（○）で示した。 (3) Quantification of + strands and − strands by qRT-PCR With respect to the sample collected by the genomic RNA replication reaction in the W / O emulsion, the quantification of + strands and − strands was performed using quantitative RT-PCR. All samples were diluted to 5 nM from the concentration determined by electrophoresis. Standard concentrations were 50, 5, and 0.5 nM. As a result, the sum of the + strand and the − strand greatly deviated from the result of quantification from electrophoresis, but the overall tendency was that the − strand was less than the + strand. FIG. 7A shows the result using the exponential phase sample, and FIG. 7B shows the result using the linear phase sample. The measured value by electrophoresis is indicated by (■), the total of qRT-PCR + and-strands is indicated by (□), the + strand of qRT-PCR is indicated by (●), and the − strand of qRT-PCR is indicated by (◯). It was.

（４）二本鎖率の測定
ゲノムＲＮＡ複製産物を電気泳動で確認した際に目的ｓｓＲＮＡの上にもう１本バンドが出る。このバンドがｄｓＲＮＡであることを過去の論文に従って確認した。＋鎖のみ５０ｎＭ、−鎖のみ５０ｎＭをアニーリングしたもの、＋鎖−鎖を１：１で混ぜて合計で５０ｎＭにしてアニーリングしたもの、それぞれ熱処理ありなしで電気泳動した。＋鎖−鎖のみのサンプルでバンドが出れば、その産物は２量体だと決定でき、混ぜたもので出れば、２本鎖（ｄｓＲＮＡ）であると決定した。次に経時変化サンプルの電気泳動写真から２本鎖率を算出した。結果、電気泳動写真から＋鎖−鎖を１：１で混ぜてアニーリングしたサンプルで、もう１本のバンドが現れたので、このバンドはｄｓＲＮＡであることが確認できた。結果を図８に示す。各実験条件は、以下のとおりである：（ｉ）＋鎖ＲＮＡ５０ｎＭ、（ｉｉ）−鎖ＲＮＡ５０ｎＭ、（ｉｉｉ）＋鎖および−鎖ＲＮＡｎ総量５０ｎＭ、（ｉｖ）反応によって生じた産物；なお、熱ショックは、９５℃ ５分間行った。 (4) Measurement of double-stranded rate When a genomic RNA replication product is confirmed by electrophoresis, another band appears on the target ssRNA. It was confirmed according to previous papers that this band was dsRNA. Electrophoresis was performed with 50 nM of the + strand alone and 50 nM of the − strand alone, and after annealing the + strand and the strand to a total of 50 nM by mixing at 1: 1. If a band appears in the + strand-strand only sample, the product can be determined to be a dimer, and if it comes out of a mixed product, it is determined to be a double strand (dsRNA). Next, the double-stranded ratio was calculated from the electrophoresis photograph of the time-varying sample. As a result, another band appeared in the sample obtained by annealing the mixture of the + strand and the strand in 1: 1 from the electrophoresis photograph, so that this band was confirmed to be dsRNA. The results are shown in FIG. Each experimental condition is as follows: (i) + strand RNA 50 nM, (ii) -strand RNA 50 nM, (iii) + strand and -strand RNAn total amount 50 nM, (iv) product produced by the reaction; The heat shock was performed at 95 ° C. for 5 minutes.

次に、タイムコースサンプルの電気泳動結果から、各時点での２本鎖率を算出した。結果、指数増幅条件（●）と線形増幅条件（○）で異なる傾向を示した。指数増幅条件では最初２本鎖が２６％と大きな値をとり、徐々に下がって最終的は１５％程度に落ち着くような結果だった。線形増幅条件では、初めから常に一定の１０％前後を示した。結果を図９に示す。ただし、２本鎖率の算出の際、初期濃度込みで計算した。 Next, the duplex rate at each time point was calculated from the electrophoresis result of the time course sample. As a result, the tendency was different between the exponential amplification condition (●) and the linear amplification condition (◯). Under the exponential amplification conditions, the first duplex had a large value of 26%, gradually decreasing, and finally settled to about 15%. Under linear amplification conditions, a constant value of around 10% was always shown from the beginning. The results are shown in FIG. However, when calculating the double-stranded ratio, it was calculated including the initial concentration.

また、本発明では、Ｈｆｑの添加により、ＲＮＡ増幅効率が促進されることも、明らかになった。 Further, in the present invention, it has also been clarified that the RNA amplification efficiency is promoted by the addition of Hfq.

本発明によって、核酸（特にＲＮＡ）増幅反応のための油中水型エマルジョンが提供される。また、本発明によって、核酸（特にＲＮＡ）増幅反応のための油中水型エマルジョンを製造する方法が提供される。 The present invention provides a water-in-oil emulsion for nucleic acid (especially RNA) amplification reactions. The present invention also provides a method for producing a water-in-oil emulsion for nucleic acid (especially RNA) amplification reactions.

配列番号１Ｑβレプリカーゼβサブユニットの核酸配列
配列番号２Ｑβレプリカーゼβサブユニットのアミノ酸配列
配列番号３Ｈｆｑタンパク質の核酸配列
配列番号４Ｈｆｑタンパク質のアミノ酸配列
配列番号５Ｓ２２２ＲＮＡの配列
配列番号６Ｑβ ｇｅｎｏｍｅｐｒｉｍｅｒ＋３の配列
配列番号７Ｑβ ｇｅｎｏｍｅｐｒｉｍｅｒ−３の配列
配列番号８ＱβレプリカーゼＥＦ−Ｔｓサブユニットの核酸配列
配列番号９ＱβレプリカーゼＥＦ−Ｔｓサブユニットのアミノ酸配列
配列番号１０ＱβレプリカーゼＥＦ−Ｔｕサブユニットの核酸配列
配列番号１１ＱβレプリカーゼＥＦ−Ｔｕサブユニットのアミノ酸配列
配列番号１２ＱβレプリカーゼＳ１サブユニットの核酸配列
配列番号１３ＱβレプリカーゼＳ１サブユニットのアミノ酸配列
SEQ ID NO: 1 Qβ replicase β subunit nucleic acid sequence SEQ ID NO: 2 Qβ replicase β subunit amino acid sequence SEQ ID NO: 3 Hfq protein nucleic acid sequence SEQ ID NO: 4 Hfq protein amino acid sequence SEQ ID NO: 5 S222 RNA sequence SEQ ID NO: 6 Qβ geneme sequence of primer + 3 SEQ ID NO: 7 sequence of Qβ genome primer-3 SEQ ID NO: 8 nucleic acid sequence of Qβ replicase EF-Ts subunit SEQ ID NO: 9 amino acid sequence of Qβ replicase EF-Ts subunit SEQ ID NO: 10 of Qβ replicase EF-Tu subunit Nucleic acid sequence SEQ ID NO: 11 Qβ replicase EF-Tu subunit amino acid sequence SEQ ID NO: 12 Qβ replicase S1 subunit nucleic acid sequence SEQ ID NO: 13 Qβ replicase S1 subunit amino acid Acid sequence

Claims

Less than:
(A) Qβ replicase;
(B) a reaction buffer; and
(C) a template nucleic acid;
A water-in-oil emulsion for nucleic acid amplification comprising an aqueous phase component containing, and an oil phase component ,
Wherein the water-in-oil emulsion is a water-in-oil emulsion prepared using an aqueous phase component and an oil phase component saturated with a buffer of the same composition as the aqueous phase component; and
The water-in-oil emulsion is
(I) a primer; and
(Ii) a solid support,
A water-in-oil emulsion that does not contain any of the above .

The water-in-oil emulsion according to claim 1, wherein the aqueous phase component comprises dithiothreitol .

The (a) Qβ replicase is:
(C1)
(C1-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2,
(C1-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 2 and consisting of the amino acid sequence shown in SEQ ID NO: 9 A polypeptide having RNA replication activity when combined with the polypeptide and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C1-3) A polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 1, comprising the amino acid sequence shown in SEQ ID NO: 9 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide comprising the amino acid sequence shown in FIG.
A polypeptide selected from the group consisting of:
(C2)
(C2-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 9,
(C2-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 9, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having RNA replication activity when combined with the polypeptide and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C2-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 8, comprising the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide comprising the amino acid sequence shown in FIG.
A polypeptide selected from the group consisting of:
(C3)
(C3-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11,
(C3-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 11, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having RNA replication activity when combined with the polypeptide and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, and
(C3-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 10, and comprising the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide consisting of the amino acid sequence shown in 9,
A polypeptide selected from the group consisting of:
The water-in-oil emulsion according to claim 1, comprising:

The (a) Qβ replicase is further
(C4)
(C4-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 13,
(C4-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 13, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 when combined with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11; A polypeptide having an RNA replication activity higher than the combination of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C4-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 12, comprising the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 when combined with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, amino acid sequence shown in SEQ ID NO: 9 2. The oil according to claim 1, comprising a polypeptide selected from the group consisting of polypeptides that exhibit higher RNA replication activity than a combination of a polypeptide consisting of and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11. Water-in-water emulsion.

The water-in-oil emulsion according to claim 1, wherein (b) the reaction buffer is an aqueous solution containing a buffer, a salt, a ribonucleotide, a reducing agent, and a protein.

The water-in-oil emulsion according to claim 1, wherein (c) the template nucleic acid has a nucleic acid sequence "CCC" at the 3 'end.

The water-in-oil emulsion according to claim 1, wherein the template nucleic acid (c) has a length of 300 bases or more.

The water-in-oil emulsion according to claim 1, wherein the water-in-oil emulsion is manufactured using a membrane having a pore size of 5 μm or more and 20 μm or less, or manufactured by vortexing.

The water-in-oil emulsion according to claim 1, further comprising (d) Hfq protein, wherein the Hfq protein is
(D1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4;
(D2) a polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 4 and promoting RNA replication activity of the Qβ replicase; and ,
(D3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 3 and that promotes the RNA replication activity of the Qβ replicase;
The water-in-oil emulsion according to claim 1, which is a polypeptide selected from the group consisting of:

The water-in-oil emulsion according to claim 1, further comprising (e) an emulsifier.

The water-in-oil emulsion according to claim 10, wherein the (e) emulsifier is selected from the group consisting of sorbitan monooleate, polyoxyethylene sorbitan monooleate, and cetyl dimethicone copolyol.

A composition for RNA replication comprising the water-in-oil emulsion according to claim 1.

A method for producing a water-in-oil emulsion for nucleic acid amplification reaction comprising the following steps:
(1) A step of preparing a mixture, the mixture comprising:
(A) an aqueous phase component,
(A) Qβ replicase,
(B) a reaction buffer, and
(C) Template nucleic acid
An aqueous phase component containing, and
(B) an oil phase component , which is saturated with a buffer having the same composition as the water phase component,
Where the mixture is
(I) a primer; and
(Ii) a solid support,
A process that does not contain any of
(2) The step of passing the mixture of the above step (1) through a membrane or the step of stirring by vortex,
Including the method.

14. The method for producing a water-in-oil emulsion according to claim 13, wherein the aqueous phase component comprises dithiothreitol .

The (a) Qβ replicase is
(C1)
(C1-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2,
(C1-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 2 and consisting of the amino acid sequence shown in SEQ ID NO: 9 A polypeptide having RNA replication activity when combined with the polypeptide and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C1-3) A polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 1, comprising the amino acid sequence shown in SEQ ID NO: 9 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide comprising the amino acid sequence shown in FIG.
A polypeptide selected from the group consisting of:
(C2)
(C2-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 9,
(C2-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 9, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having RNA replication activity when combined with the polypeptide and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C2-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 8, comprising the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide comprising the amino acid sequence shown in FIG.
A polypeptide selected from the group consisting of:
(C3)
(C3-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11,
(C3-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 11, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide having RNA replication activity when combined with the polypeptide and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9, and
(C3-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 10, and comprising the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: A polypeptide having RNA replication activity when combined with a polypeptide consisting of the amino acid sequence shown in 9,
A polypeptide selected from the group consisting of:
The method for producing a water-in-oil emulsion according to claim 13, comprising:

The (a) Qβ replicase is further
(C4)
(C4-1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 13,
(C4-2) A polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 13, and consisting of the amino acid sequence shown in SEQ ID NO: 2 A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 when combined with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11; A polypeptide having an RNA replication activity higher than the combination of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 and the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and
(C4-3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 12, comprising the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 when combined with a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, amino acid sequence shown in SEQ ID NO: 9 The oil according to claim 13, comprising a polypeptide selected from the group consisting of polypeptides that exhibit higher RNA replication activity than a combination of a polypeptide consisting of and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11. Water-in-water emulsion production method.

The method for producing a water-in-oil emulsion according to claim 13, wherein the reaction buffer (b) is an aqueous solution containing a buffer, a salt, a ribonucleotide, a reducing agent, and a protein.

The method for producing a water-in-oil emulsion according to claim 13, wherein the template nucleic acid (c) is a nucleic acid having a nucleic acid sequence "CCC" at the 3 'end.

The method for producing a water-in-oil emulsion according to claim 13, wherein the template nucleic acid (c) has a length of 300 bases or more.

The method for producing a water-in-oil emulsion according to claim 13, wherein the pore size diameter of the membrane is 5 µm or more and 20 µm or less.

14. The method of producing a water-in-oil emulsion according to claim 13, wherein the mixture of step (1) further comprises (d) Hfq protein, wherein the Hfq protein is
(D1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4;
(D2) a polypeptide comprising an amino acid sequence containing one or several deletions, additions or substitutions in the amino acid sequence shown in SEQ ID NO: 4 and promoting RNA replication activity of the Qβ replicase; and ,
(D3) a polypeptide encoded by a nucleic acid that hybridizes under stringent conditions to the nucleic acid sequence shown in SEQ ID NO: 3 and that promotes the RNA replication activity of the Qβ replicase;
A method for producing a water-in-oil emulsion, which is a polypeptide selected from the group consisting of:

The method for producing a water-in-oil emulsion according to claim 13, wherein the mixture of the step (1) further comprises (e) an emulsifier.

23. The method for producing a water-in-oil emulsion according to claim 22, wherein the emulsifier (e) is selected from the group consisting of sorbitan monooleate, polyoxyethylene sorbitan monooleate, and cetyl dimethicone copolyol.