JP6921478B2 - Transcription / translation conjugated cell-free protein synthesis - Google Patents
Transcription / translation conjugated cell-free protein synthesis Download PDFInfo
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- JP6921478B2 JP6921478B2 JP2016015070A JP2016015070A JP6921478B2 JP 6921478 B2 JP6921478 B2 JP 6921478B2 JP 2016015070 A JP2016015070 A JP 2016015070A JP 2016015070 A JP2016015070 A JP 2016015070A JP 6921478 B2 JP6921478 B2 JP 6921478B2
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Description
本発明は、転写・翻訳共役型無細胞系タンパク質合成に使用するDNA断片、反応液、鋳型DNA、タンパク質の製造方法に関する。 The present invention relates to a method for producing a DNA fragment, a reaction solution, a template DNA, and a protein used for transcription / translation-conjugated cell-free protein synthesis.
近年、ヒトゲノムを始め多くの生物の遺伝情報が解読されてきている。このような中、ポストゲノム研究として、これらの遺伝情報に対応するタンパク質の機能解析やゲノム創薬が注目を集めている。これらの遺伝情報に対応するタンパク質を医薬品などに応用、利用するには、莫大な種類のタンパク質を短時間で簡単に合成することが必要となってくる。 In recent years, the genetic information of many organisms including the human genome has been deciphered. Under these circumstances, functional analysis of proteins corresponding to these genetic information and genomic drug discovery are attracting attention as post-genome research. In order to apply and use proteins corresponding to these genetic information in pharmaceutical products, it is necessary to easily synthesize a huge variety of proteins in a short time.
現在、タンパク質の生産方法には、遺伝子組換え技術によって酵母や昆虫細胞（昆虫培養細胞）などの生細胞を用いる発現系（以下、「細胞系」ということがある）が広く利用されている。しかし、生細胞は自己機能を維持するために外来タンパク質を排除する傾向があり、また生細胞で細胞毒タンパク質を発現すると細胞が生育しないなど発現が困難なタンパク質も多い。 Currently, as a protein production method, an expression system (hereinafter, sometimes referred to as "cell line") using live cells such as yeast and insect cells (insect cultured cells) by gene recombination technology is widely used. However, living cells tend to eliminate foreign proteins in order to maintain their own functions, and when cytotoxic proteins are expressed in living cells, many proteins are difficult to express, such as cells not growing.
一方、細胞系を使用しないタンパク質の生産方法として、細胞破砕液や抽出液に基質や酵素などを加えるなどして生物の遺伝情報翻訳系または遺伝情報転写／翻訳系を試験管内に取り揃え、反応鋳型として目的タンパク質をコードする構造遺伝子を有するＤＮＡ（転写鋳型）またはｍＲＮＡ（翻訳鋳型）を用いて、アミノ酸を望みの順番に必要な残基数結合させることのできる合成系を再構築する、無細胞系のタンパク質合成が知られている。このような無細胞系タンパク質合成では、上記細胞系のタンパク質合成のような制約を受けにくく、生物の命を断つことなくタンパク質の合成を行うことができ、またタンパク質の生産に培養などの操作を伴わないため細胞系と比較して短時間にタンパク質の合成を行うことができる。 On the other hand, as a protein production method that does not use a cell system, a genetic information translation system or a genetic information transcription / translation system of an organism is prepared in vitro by adding a substrate or an enzyme to a cell disruption solution or an extract, and a reaction template is provided. Using DNA (transcription template) or mRNA (translation template) having a structural gene encoding the protein of interest, a cell-free system is reconstructed that can bind amino acids in the desired order by the required number of residues. The protein synthesis of the system is known. In such cell-free protein synthesis, it is less likely to be restricted by the above-mentioned cell-based protein synthesis, protein synthesis can be performed without killing the life of the organism, and operations such as culture are performed for protein production. Since it is not involved, protein synthesis can be performed in a shorter time than in a cell system.
特許文献１〜４は、転写と翻訳を別々の容器で独立して行う無細胞系タンパク質合成系の発現効率を向上させるDNA断片等を開示する。 Patent Documents 1 to 4 disclose DNA fragments and the like that improve the expression efficiency of a cell-free protein synthesis system in which transcription and translation are performed independently in separate containers.
無細胞系タンパク質合成において、転写と翻訳で好ましい反応条件、特にMg濃度が異なるため転写と翻訳を別の反応容器で行う転写・翻訳独立型の反応が行われていたが、この方法は操作が煩雑であり、改善が求められていた。 In cell-free protein synthesis, a transcription / translation independent reaction in which transcription and translation are performed in different reaction vessels because the favorable reaction conditions for transcription and translation, especially Mg concentration, are different, but this method can be operated. It was complicated and required improvement.
無細胞タンパク質合成系において、転写と翻訳を１つの容器内で共役して行うことは可能であるが、転写と翻訳に適した反応条件は異なるため、タンパク質の発現効率が低下するため、現在はほとんど採用されていない。 In a cell-free protein synthesis system, transcription and translation can be coupled in one container, but the reaction conditions suitable for transcription and translation are different, and the protein expression efficiency is reduced. Almost not adopted.
大腸菌、酵母、真核生物細胞などを用いたタンパク質合成系は、１つのタンパク質を大量に合成するのには都合がよいが、多数のタンパク質を少量ずつ合成する場合には無細胞タンパク質合成系が適している。 A protein synthesis system using Escherichia coli, yeast, eukaryotic cells, etc. is convenient for synthesizing a large amount of one protein, but a cell-free protein synthesis system is suitable for synthesizing a large number of proteins little by little. Are suitable.
本発明は、タンパク質の発現効率の高い無細胞タンパク質合成技術を提供することを目的とする。 An object of the present invention is to provide a cell-free protein synthesis technique having high protein expression efficiency.
本発明者は ABPV(Acute bee paralysis virus) IGR(intergenic region)-IRES(internal ribosome entry sites)は転写・翻訳独立型の無細胞系では公知のAcNPV 5’UTRよりも効率が劣るが、転写・翻訳共役型の無細胞系では飛躍的に発現効率が高くなることを見出した。 The present inventor found that ABPV (Acute bee paralysis virus) IGR (intergenic region) -IRES (internal ribosome entry sites) is less efficient than the known AcNPV 5'UTR in a transcription / translation independent cell-free system, but transcription / translation It was found that the expression efficiency is dramatically increased in the translation-conjugated cell-free system.
本発明は、転写・翻訳共役型無細胞系タンパク質合成系におけるABPV IGR-IRESの使用、鋳型ＤＮＡ、反応液及びタンパク質の製造方法を提供するものである。
項１． 転写・翻訳共役型の無細胞タンパク質合成系においてタンパク質の発現量を増大させるためのABPV(Acute bee paralysis virus) IGR(intergenic region)-IRES(internal ribosome entry sites)核酸配列の使用。
項２． タンパク質をコードする構造遺伝子がABPV IGR-IRESの制御下に組み込まれた転写・翻訳共役型の無細胞タンパク質合成用鋳型ＤＮＡ。
項３． 項２に記載の鋳型ＤＮＡ及びリボソームを含有する転写・翻訳共役型の無細胞タンパク質合成用反応液。
項４． カイコガ抽出液をさらに含む、項３に記載の反応液。
項５． 項３又は４に記載の反応液を用いて構造遺伝子の転写と翻訳を１つの容器内で行い目的とするタンパク質を得ることを特徴とする、転写・翻訳共役型の無細胞タンパク質合成系におけるタンパク質の製造方法。
The present invention provides the use of ABPV IGR-IRES in a transcription / translation-conjugated cell-free protein synthesis system, and a method for producing a template DNA, a reaction solution, and a protein.
Item 1. Use of ABPV (Acute bee paralysis virus) IGR (intergenic region) -IRES (internal ribosome entry sites) nucleic acid sequences to increase protein expression in transcriptional / translation-conjugated cell-free protein synthesis systems.
Item 2. A transcription / translation-conjugated template DNA for cell-free protein synthesis in which a structural gene encoding a protein is integrated under the control of ABPV IGR-IRES.
Item 4.
本発明によれば、１つの反応容器で転写と翻訳を同時に進行させる転写・翻訳共役型の無細胞タンパク質合成系を用いて高効率で目的タンパク質を合成することができる。 According to the present invention, a target protein can be synthesized with high efficiency by using a transcription / translation-coupled cell-free protein synthesis system in which transcription and translation proceed simultaneously in one reaction vessel.
本発明は、構造遺伝子をABPV IGR-IRESの制御下に置いたDNA断片を転写・翻訳共役型の無細胞系において発現させる。好ましいDNA断片は構造遺伝子の発現ベクターであり、例えばプラスミドを使用することができる。 The present invention expresses a DNA fragment in which a structural gene is placed under the control of ABPV IGR-IRES in a transcription / translation-conjugated cell-free system. A preferred DNA fragment is an expression vector for a structural gene, for example a plasmid can be used.
ABPV IGR-IRESは、GenBankのアクセッション番号がAF150629の6301-6569のDNA配列であり、その配列を配列番号1及び図７に示す。 ABPV IGR-IRES is the DNA sequence of 6301-6569 of GenBank accession number AF150629, and the sequence is shown in SEQ ID NO: 1 and FIG.
ABPV IGR-IRESは、構造遺伝子を発現させる機能を有する限り、1又は複数個、例えば１〜40個、好ましくは１〜30個、より好ましくは１〜25個、さらに好ましくは1〜20個、特に1〜15個の塩基が置換、付加、欠失又は挿入されていてもよい。また、構造遺伝子は、構造遺伝子を発現させる機能を有する限り、80%以上、好ましくは85％以上、より好ましくは90％以上、さらに好ましくは95％以上、特に98％以上の同一性を有する塩基配列であってもよい。ABPV IGR-IRESの制御下に構造遺伝子を挿入し、転写・翻訳共役型の無細胞系で発現させることにより、構造遺伝子の発現産物であるタンパク質を製造することができる。得られたタンパク質は、再沈殿、カラムクロマトグラフィー、透析などの常法の精製手段に従い精製することができる。 ABPV IGR-IRES may be one or more, for example 1 to 40, preferably 1 to 30, more preferably 1 to 25, even more preferably 1 to 20, as long as it has the function of expressing a structural gene. In particular, 1 to 15 bases may be substituted, added, deleted or inserted. Further, the structural gene is a base having 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly 98% or more identity as long as it has a function of expressing the structural gene. It may be an array. By inserting a structural gene under the control of ABPV IGR-IRES and expressing it in a transcription / translation-conjugated cell-free system, a protein that is an expression product of the structural gene can be produced. The resulting protein can be purified according to conventional purification means such as reprecipitation, column chromatography and dialysis.
ABPV IGR-IRESの5’側から、25個以下、好ましくは、20個以下、より好ましくは15個以下、さらに好ましくは10個以下、特に5個以下の塩基を欠失させることができる。また、ABPV IGR-IRESの3’側から、30個以下、好ましくは25個以下、より好ましくは、20個以下、さらに好ましくは15個以下、特に好ましくは10個以下、最も好ましくは5個以下の塩基を欠失させることができる。 From the 5'side of ABPV IGR-IRES, 25 or less, preferably 20 or less, more preferably 15 or less, still more preferably 10 or less, particularly 5 or less bases can be deleted. From the 3'side of ABPV IGR-IRES, 30 or less, preferably 25 or less, more preferably 20 or less, still more preferably 15 or less, particularly preferably 10 or less, most preferably 5 or less. Can be deleted from the base of.
本発明のDNA断片は、ABPV IGR-IRESの5’上流側にプロモーターを有することが好ましい。プロモーターとしては、通常のものが制限なく使用することができ、例えばT7プロモーター、SP6プロモーター、T3プロモーターなどが挙げられる。また、DNA断片の構造遺伝子の下流側に3’非翻訳領域及びポリＡ配列、ターミネーター配列を有していてもよい。 The DNA fragment of the present invention preferably has a promoter 5'upstream of ABPV IGR-IRES. As the promoter, ordinary promoters can be used without limitation, and examples thereof include a T7 promoter, an SP6 promoter, and a T3 promoter. Further, it may have a 3'untranslated region, a poly A sequence, and a terminator sequence on the downstream side of the structural gene of the DNA fragment.
構造遺伝子は、精製を容易にするためにHisタグ、GSTタグなどを連結してもよい。構造遺伝子がコードするタンパク質は特に限定されないが、各種の酵素、ホルモン、サイトカイン、医薬、抗原、抗体、受容体などの有用タンパク質が挙げられる。アミノ酸配列が少し異なる多数のタンパク質を各々の転写・翻訳共役型の無細胞発現系で発現させてもよい。 The structural gene may be linked with a His tag, a GST tag, or the like to facilitate purification. The protein encoded by the structural gene is not particularly limited, and examples thereof include useful proteins such as various enzymes, hormones, cytokines, drugs, antigens, antibodies, and receptors. A large number of proteins having slightly different amino acid sequences may be expressed in each transcription / translation-conjugated cell-free expression system.
転写・翻訳共役型の無細胞系タンパク質合成に使用する抽出物としては、小麦胚芽などの植物由来抽出物、ウサギ網状赤血球などの哺乳動物由来の抽出物、大腸菌などの細菌由来の抽出物、カイコガなどの昆虫培養細胞由来の抽出物のいずれを用いてもよいが、昆虫由来の抽出物が好ましく、カイコガ由来の抽出物が最も好ましい。 Extracts used for transcription / translation-conjugated cell-free protein synthesis include plant-derived extracts such as wheat germ, mammalian-derived extracts such as rabbit reticular erythrocytes, bacterial-derived extracts such as Escherichia coli, and silkworm. Any of the extracts derived from cultured insect cells such as the above may be used, but the extract derived from insects is preferable, and the extract derived from silkworm mosquito is most preferable.
転写・翻訳共役型の無細胞系の反応液には、ABPV IGR-IRESの制御下に構造遺伝子を含むDNA断片（例えばプラスミド）、マグネシウムイオン、カリウムイオン、NTP、DTT（ジチオスレイトール、任意成分）、アミノ酸(任意成分)、スペルミジン、RNAポリメラーゼ、転写・翻訳を可能にする細胞由来の抽出物(lysate)などが含まれる。構造遺伝子のコドンユーセージは、これらの抽出物に適したものに改変することが好ましい。 Transcription / translation-conjugated cell-free reaction fluid contains DNA fragments (eg plasmids) containing structural genes under the control of ABPV IGR-IRES, magnesium ions, potassium ions, NTP, DTT (dithiothreitol, optional components). ), Amino acid (optional component), spermidine, RNA polymerase, cell-derived extract (lysate) that enables transcription and translation, etc. The codon usage of the structural gene is preferably modified to be suitable for these extracts.
マグネシウムイオンは、酢酸マグネシウム、塩化マグネシウムなどのマグネシウム塩として反応液に添加することができる。好ましくは酢酸マグネシウムが挙げられる。マグネシウムイオンの濃度としては、無細胞系でタンパク質が生産できる限り特に限定されないが、好ましくは1.6mM〜1.8mM程度が挙げられる。 Magnesium ions can be added to the reaction solution as magnesium salts such as magnesium acetate and magnesium chloride. Magnesium acetate is preferable. The concentration of magnesium ions is not particularly limited as long as the protein can be produced in a cell-free system, but is preferably about 1.6 mM to 1.8 mM.
カリウムイオンは、酢酸カリウム、塩化カリウムなどのカリウム塩として反応液に添加することができる。好ましくは酢酸カリウムが挙げられる。カリウムイオンの濃度としては、無細胞系でタンパク質が生産できる限り特に限定されないが、好ましくは100mM〜150mM程度が挙げられる。 Potassium ions can be added to the reaction solution as potassium salts such as potassium acetate and potassium chloride. Potassium acetate is preferable. The concentration of potassium ions is not particularly limited as long as the protein can be produced in a cell-free system, but preferably about 100 mM to 150 mM.
NTP（ATP, GTP, UTP, CTP）の濃度としては、0.2mM〜0.6mM程度が挙げられる。 The concentration of NTP (ATP, GTP, UTP, CTP) is about 0.2 mM to 0.6 mM.
DTTはなくても問題ないが、例えば0〜0.05mM程度で使用してもよい。 There is no problem even if there is no DTT, but for example, it may be used at about 0 to 0.05 mM.
カイコガなどの抽出液(lysate)としては、例えば10〜20%(v/v)程度が挙げられる。 Examples of the extract (lysate) of silk moth and the like include about 10 to 20% (v / v).
アミノ酸は、添加しなくてもよいが、反応液1ml当たり0〜60μM程度添加してもよい。 Amino acids may not be added, but may be added in an amount of about 0 to 60 μM per 1 ml of the reaction solution.
スペルミジンは、例えば0.1〜1mM程度、好ましくは0.2〜0.3mM程度添加する。 Spermidine is added, for example, about 0.1 to 1 mM, preferably about 0.2 to 0.3 mM.
転写・翻訳共役型無細胞系タンパク質合成系の反応温度は、15〜32℃程度、好ましくは25〜30℃程度、最も好ましくは28℃程度である。このような反応温度で合成されるタンパク質量が多くなる。 The reaction temperature of the transcription / translation-conjugated cell-free protein synthesis system is about 15 to 32 ° C, preferably about 25 to 30 ° C, and most preferably about 28 ° C. The amount of protein synthesized at such a reaction temperature increases.
転写・翻訳共役型無細胞系タンパク質合成系のpHは、6〜8程度、好ましくは7〜7.8程度である。 The pH of the transcription / translation-conjugated cell-free protein synthesis system is about 6 to 8, preferably about 7 to 7.8.
以下、実施例及び比較例によって本発明をより詳細に説明するが、本発明は実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to the Examples.
実施例及び比較例
1. カイコガの飼育と後部絹糸腺の摘出
1-1. カイコガの飼育
実験には錦秋×鐘和（上田蚕種）を用い，飼育は松原ら（1988）の方法に従い, 人工飼料はKIT-512 （無菌養蚕研究所）を用い，26±1°Cに設定されたクリーンルーム内での完全無菌人工飼料飼育を行った。
Examples and Comparative Examples
1. Breeding of silk moths and removal of posterior silk glands
1-1. Silk moth breeding experiment using Kinshu x Kanewa (Ueda silkworm species), breeding according to the method of Matsubara et al. (1988), artificial feed using KIT-512 (sterile silkworm research institute), 26 ± Completely sterile artificial feed was bred in a clean room set at 1 ° C.
実験に供試したカイコは発達段階を同調させるために，4齢0日でいったん整え，5齢0日においては6時間以内に5齢脱皮を完了した個体を集めた。 In order to synchronize the developmental stages, the silk moths used in the experiment were once prepared at 4th instar 0 days, and at 5th instar 0 days, individuals who completed 5th instar molting within 6 hours were collected.
1-2. 後部絹糸腺の摘出
5齢4日の幼虫を氷冷した 0.75% NaCl 溶液中で背側から切開し, 中腸を除いた後, 絹糸腺のうち, 後部絹糸腺のみを摘出した。 摘出された器官は, 別の容器に準備しておいた氷冷0.75% NaCl溶液中で, 器官などの付着物を取り除いたのち, ろ紙で充分に水分を取り除き, 1.5 g ずつアルミホイルで包み, 液体窒素で凍結した後, 実験に使用するまで-80°C下で保存した。
1-2. Removal of the posterior silk gland
The 5th instar 4 day larvae were incised from the dorsal side in an ice-cooled 0.75% NaCl solution, the midgut was removed, and only the posterior silk gland was removed. The removed organs were placed in an ice-cooled 0.75% NaCl solution prepared in a separate container to remove deposits such as the organs, and then sufficiently removed with filter paper and wrapped in aluminum foil in 1.5 g portions. After freezing in liquid nitrogen, it was stored at -80 ° C until it was used in the experiment.
2. プラスミド
2-1. プラスミドの構築
2-1-1. pGEM-LA （IRESを含まないプラスミド）
pGL3-Control Vector (Promega)を鋳型とし，ホタルルシフェラーゼ遺伝子をpolymerase chain reaction (PCR) により増幅した。ホタルルシフェラーゼ遺伝子はpGL3-Control Vector の88 ‐1737領域にコードされている。そこで，88 以降に相当する配列にEcoR Iサイトを付加した TNT-Luc#1 (5’ -GGAATTCATGGAAGACGCCAAAAACATAAAGAAAGG- 3’ ) と，1737までに相当する配列にSal Iサイトを付加した TNT-Luc#2 (5’ -ACGCGTCGACTTACACGGCGATCTTTCCGCCCTTC- 3’ ) をプライマーとして，Pyrobest DNA polymerase (TAKARA Bio) を用い，98℃, 10秒，61℃, 30秒，72℃, 60秒 を1サイクルとして25回の反応を行い，増幅されたDNAはNucleoSpin Extract II (MACHEREY-NAGEL) で精製した。精製DNAとpTNT Vector (Promega) は制限酵素, EcoR I,とSal Iでそれぞれ消化し，ライゲーション反応により生成されたプラスミドをpTNT-Lucと名づけた。
2. plasmid
2-1. Construction of plasmid
2-1-1. PGEM-LA (IRES-free plasmid)
Using the pGL3-Control Vector (Promega) as a template, the firefly luciferase gene was amplified by the polymerase chain reaction (PCR). The firefly luciferase gene is encoded in the 88-1737 region of the pGL3-Control Vector. Therefore, 88 after EcoR the sequences corresponding to the I T N T-Luc was added Site # 1 and (5 '-G GAATTC ATGGAAGACGCCAAAAACATAAAGAAAGG- 3' ), T was added to Sal I site in the sequence corresponding to the up 1737 N Using T-Luc # 2 (5'-ACGC GTCGAC TTACACGGCGATCTTTCCGCCCTTC-3') as a primer and using Pyrobest DNA polymerase (TAKARA Bio), 98 ° C, 10 seconds, 61 ° C, 30 seconds, 72 ° C, 60 seconds for one cycle. The reaction was carried out 25 times, and the amplified DNA was purified with Nucleo Spin Extract II (MACHEREY-NAGEL). The purified DNA and pT N T Vector (Promega) were digested with restriction enzymes, EcoR I, and Sal I, respectively, and the plasmid produced by the ligation reaction was named pT N T-Luc.
次に，pTNT-Lucを制限酵素 ，EcoR IとBamH Iで消化し，これをTAE [0.04M Tris，0.04M Acetic acid，0.5M EDTA (pH 8.0)] を含む1 % Agarose ゲル (Seakem GTG：BMA) をTAE緩衝液，50V定電圧で電気泳動した．EtBr (エチレンブロマイド) で染色後，UVイルミネーター上で 1,744 bpのDNA断片をゲルから切り出した．切り出したゲルはNucleoSpin Extract II (MACHEREY-NAGEL)を用い， DNAの精製，回収を行った．これを，制限酵素 ，EcoR I,とBamH Iで消化したpGEM-3Zf (+) Vector (Promega)とライゲーション反応をおこない，生成されたプラスミドベクターをpGEM-LAと名づけた。 Next, restriction enzymes pT N T-Luc, was digested with EcoR I and BamH I, which TAE 1% Agarose gel (Seakem containing [0.04M Tris, 0.04M Acetic acid, 0.5M EDTA (pH 8.0)] GTG: BMA) was electrophoresed in TAE buffer at a constant voltage of 50 V. After staining with EtBr (ethylene bromide), a 1,744 bp DNA fragment was excised from the gel on a UV illuminator. The excised gel was purified and recovered from DNA using Nucleo Spin Extract II (MACHEREY-NAGEL). This was ligated with pGEM-3Zf (+) Vector (Promega) digested with restriction enzymes EcoR I and Bam H I, and the resulting plasmid vector was named pGEM-LA.
2-1-2. pAc-LA(比較例で使用する公知のプラスミド)
pTNT-Lucを制限酵素Bgl IIとXho Iで消化し，この間に，T7 RNA polymarase promotor (TAATACGACTCACTATAGG) と夜盗蛾の一種 (Autographa californica)の 核多核体病ウイルス (nuclear polyhdrosis virus) のポリヘドリン遺伝子の5’ UTR配列(5’-AAGTATTTTACTGTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAAT-3’; Possee et al., 1991) を挿入した。
2-1-2. PAc-LA (known plasmid used in Comparative Examples)
was digested pT N T-Luc with restriction enzymes Bgl II and Xho I, during which the polyhedrin gene of T7 RNA polymarase promotor (TAATACGACTCACTATAGG) a type of cabbage armyworm (Autographa californica) of the nuclear polyhedrosis virus (nuclear polyhdrosis virus) 5'UTR sequence (5'-AAGTATTTTACTGTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAAT-3'; Possee et al., 1991) was inserted.
2-1-3. pPSIV-LA
Plautia stali intestine virus (PSIV; GenBank Accession No. AB006531) の複製酵素前駆体遺伝子と外皮タンパク質前駆体遺伝子の間にあるIGR-IRES配列およびその前後の配列がクローニングされたpT7CAT5373 (平塚和之 教授, 横浜国立大学より分与された) を鋳型として，5,955以降に相当する配列にBgl IIサイト(AGATCT)，RNA polymarase promotor配列を付加した PSIV2-1 (5’ -GAAGATCTAATACGACTCACTATAGGACACGCGGCCTTCCAAGCAGTTAG- 3’ ) と，6,223までに相当する配列にXho Iサイトを付加した PSIV2-2 (5’ -CCGCTCGAGCCTGCCTGCGCTCCTGGTATACGC- 3’ ) をプライマーとして，Pyrobest DNA polymerase (TAKARA Bio) を用い，98℃ 10秒，63℃ 30秒，72℃ 60秒 を1サイクルとして30回の反応を行った。この増幅されたDNAを精製した後，pTNT-LucのBgl II, Xho Iサイトに挿入した。
2-1-3. pPSIV-LA
The IGR-IRES sequence between the replication enzyme precursor gene and the coat protein precursor gene of Plautia stali intestine virus (PSIV; GenBank Accession No. AB006531) and the sequences before and after it were cloned into pT7CAT5373 (Professor Kazuyuki Hiratsuka, Yokohama). PSIV2-1 (5'-GA AGATCTAATACGACTCACTATAGG ACACGCGGCCTTCCAAGCAGTTAG-3') and 6,223 PSIV2-2 (5'-CCGCTCGAGCCTGCCTGCGCTCCT GGTATA CGC-3') with Xho I site added to the sequence corresponding to the above was used as a primer, using Pyrobest DNA polymerase (TAKARA Bio), 98 ° C for 10 seconds, 63 ° C for 30 seconds, The reaction was carried out 30 times with a cycle of 72 ° C for 60 seconds. After purification of the amplified DNA, was inserted into the Bgl II, Xho I sites of pT N T-Luc.
2-1-4. pCrPV-LA, pDCV-LA, pRhPV-LA, pABPV-LA
Cricket paralysis virus isolate CrPV-3 (CrPV; GenBank Accession No. KP974707), Drosophila C virus strain EB (DCV; GenBank Accession No. AF014388), Rhopalosiphum padi virus (RhPV ; GenBank Accession No. AF022937), Acute bee paralysis virus (ABPV; GenBank Accession No. AF150629),のそれぞれIGR-IRES配列およびその前後の配列(Nakajima and Uchiumi, 2009 ; 5,980-6,248, 6,026-6,296, 6,885-7,141, 6,301-6,569) に，5’端にはBgl II, T7 promoter配列 (AGATCTAATACGACTCACTATAGG) を, 3’端にはXho I配列 (CTCGAG) を附加した配列を化学合成し, pUC119に由来するBlue Heron pUC; BLUEHERON Biotechnology) にクローニングしたpUC-CrPV, pUC-DCV, pUC-RhPV, pUC-ABPV,をLife Technologiesに依頼して作成した。各配列を図7に示す。
2-1-4. pCrPV-LA, pDCV-LA, pRhPV-LA, pABPV-LA
Cricket paralysis virus isolate CrPV-3 (CrPV; GenBank Accession No. KP974707), Drosophila C virus strain EB (DCV; GenBank Accession No. AF014388), Rhopalosiphum padi virus (RhPV; GenBank Accession No. AF022937), Acute bee paralysis virus ( ABPV; GenBank Accession No. AF150629), respectively, in the IGR-IRES sequence and the sequences before and after it (Nakajima and Uchiumi, 2009; 5,980-6,248, 6,026-6,296, 6,885-7,141, 6,301-6,569), at the 5'end. PUC-CrPV, pUC cloned into Blue Heron pUC; BLUEHERON Biotechnology) derived from pUC119 by chemically synthesizing the Bgl II, T7 promoter sequence (AGATTCTAATACGACTCACTATAGG) and the sequence with the Xho I sequence (CTCGAG) at the 3'end. -DCV, pUC-RhPV, pUC-ABPV, were created by requesting Life Technologies. Each sequence is shown in FIG.
すべてのプラスミドは，全て大腸菌DH5αに形質転換し, 50 ppm Ampicilinを含む Luria-Bertanis broth (LB) 50 ml で37°C, 18 時間培養した。 このプラスミドを精製した後, Bgl II, Xho Iで消化した。 これをTAE [0.04 M Tris，0.04 M Acetic acid，0.5 M EDTA (pH 8.0)] を含む1 % Agarose ゲル (Seakem GTG：BMA) を用いてTAE緩衝液，50V定電圧で電気泳動した。 EtBr (エチジウムブロマイド) で染色後，化学合成により作製したDNA部分に相当する300 bp付近のDNA断片をゲルから切り出した。切り出したゲルからキットの説明書に従いNucleoSpin Extract II (MACHEREY-NAGEL) を用いて，DNAの精製，回収を行った。次に，それぞれのIGR-IRESをpTNT-LucのBgl II, Xho Iサイトに挿入した。結果，生成されたCrPV, DCV, RhPV, ABPVのIGR-IRESを含むプラスミドを, それぞれpCrPV-LA, pDCV-LA, pRhPV-LA, pABPV-LAと名付けた。 All plasmids were transformed into E. coli DH5α and cultured in 50 ml of Luria-Bertanis broth (LB) containing 50 ppm Ampicilin at 37 ° C for 18 hours. After purifying this plasmid, it was digested with Bgl II and Xho I. This was electrophoresed in TAE buffer at a constant voltage of 50 V using a 1% Agarose gel (Seakem GTG: BMA) containing TAE [0.04 M Tris, 0.04 M Acetic acid, 0.5 M EDTA (pH 8.0)]. After staining with EtBr (ethidium bromide), a DNA fragment of about 300 bp corresponding to the DNA portion prepared by chemical synthesis was cut out from the gel. DNA was purified and recovered from the cut gel using Nucleo Spin Extract II (MACHEREY-NAGEL) according to the instructions of the kit. Next, each IGR-IRES was inserted into the Bgl II and Xho I sites of pT N T-Luc. As a result, the generated plasmids containing IGR-IRES of CrPV, DCV, RhPV, and ABPV were named pCrPV-LA, pDCV-LA, pRhPV-LA, and pABPV-LA, respectively.
2-1-5. pABPV-LAの短縮化および改変
第2-1-4項で構築したpABPV-LAを鋳型に，表１に表すプライマーを用い，KOD -plus- Mutagenesis Kit (TOYOBO) を使用して，配列の短縮化および改変をおこなった。なお，PCR条件は94 °C 2分, (98 °C 10秒, 68 °C 7分10秒)×5回, または10回の条件でPCRを行った。なお，配列の短縮化および改変をシークエンスにより確認した。
2-1-5. Shortening and modification of pABPV-LA Using the pABPV-LA constructed in Section 2-1-4 as a template, the primers shown in Table 1 were used, and the KOD -plus- Mutagenesis Kit (TOYOBO) was used. Then, the sequence was shortened and modified. The PCR conditions were 94 ° C for 2 minutes, (98 ° C for 10 seconds, 68 ° C for 7 minutes and 10 seconds) x 5 times, or 10 times. The shortening and modification of the sequence were confirmed by sequence.
2-2. プラスミドDNAの調製
上記すべてのプラスミドは大腸菌DH5αに形質転換し, 50 ppm Ampicilinを含む Luria-Bertanis broth (LB) 50 ml で37°C, 18時間培養した。これをPure Yield Midiprep System (Promega) を用い, キットの説明に従って, プラスミドを精製した。なお，精製したプラスミドは適当に希釈し，Qubit 2.0 Fluorometer (Life Technologies) を用いて，添付された説明書に従ってプラスミドDNAの純度及び濃度を求めた。
2-2. Preparation of plasmid DNA All the above plasmids were transformed into Escherichia coli DH5α and cultured in 50 ml of Luria-Bertanis broth (LB) containing 50 ppm Ampicilin at 37 ° C for 18 hours. The plasmid was purified using the Pure Yield Midiprep System (Promega) according to the instructions in the kit. The purified plasmid was appropriately diluted, and the purity and concentration of the plasmid DNA were determined using Qubit 2.0 Fluorometer (Life Technologies) according to the attached instructions.
3. 無細胞翻訳反応系
3-1. in vitro transcriptionによるRNA合成
前項で構築・精製したプラスミドDNAを BamH I で切断して直鎖化したのち, フェノール/クロロホルム処理を2回, クロロホルム処理を1回行い, エタノール沈殿によりDNA を沈殿させ, 蒸留水に再溶解した。
3. Cell-free translation reaction system
3-1. RNA synthesis by in vitro transcription After cutting the plasmid DNA constructed and purified in the previous section with BamHI to linearize it, perform phenol / chloroform treatment twice and chloroform treatment once, and DNA by ethanol precipitation. Was precipitated and redissolved in distilled water.
この直鎖化したDNAを鋳型として， Ribomax Large Scale RNA Production System - T7 (Promega) を用い, キットに添付されている説明書に従って, in vitro transcription によりRNA を合成した。 反応時間は4時間とし, 反応終了後, RNase-free DNaseで処理し, フェノール/クロロホルム処理を2回, クロロホルム処理を1回行い, エタノール沈殿法により, RNAを沈殿させた。 その後, 全量を100 μlのD.W. に溶解し, NICK columns (GE helthcare) に供してヌクレオチドの排除を目的とした精製を行った。精製したRNAは適当に希釈し，添付された説明書に従い，Qubit 2.0 Fluorometer (Life Technologies)を用いて濃度を求め，最終的に全て2.0 μg/μlになるように蒸留水で調整した。なお，以降の無細胞翻訳反応系には，-80°Cに少量ずつ分注・保存したものを使用した。 Using this linearized DNA as a template, RNA was synthesized by in vitro transcription using the Ribomax Large Scale RNA Production System --T7 (Promega) according to the instructions attached to the kit. The reaction time was 4 hours. After completion of the reaction, RNase-free DNase treatment was performed, phenol / chloroform treatment was performed twice, and chloroform treatment was performed once, and RNA was precipitated by the ethanol precipitation method. Then, the whole amount was dissolved in 100 μl of D.W. and subjected to NICK columns (GE helthcare) for purification for the elimination of nucleotides. The purified RNA was appropriately diluted, the concentration was determined using a Qubit 2.0 Fluorometer (Life Technologies) according to the attached instructions, and the final concentration was adjusted with distilled water so as to be 2.0 μg / μl. For the subsequent cell-free translation reaction system, those that were dispensed and stored in small amounts at -80 ° C were used.
3-2. 器官抽出液の作製
凍結状態の後部絹糸腺を, 氷冷した0.75% NaCl 溶液に移し, 穏やかに解凍した。完全に解凍が確認された後, 器官に付着する余分な水分をろ紙で取り除き, これを, あらかじめ氷冷したLoose タイプのガラス製ダウンス型ホモジナイザー （直径 13 mm, 長さ 82 mm, 筒長175 mm, Wheaton）に移した。 後部絹糸腺 1.5 g に対して氷冷した抽出緩衝液 [20 mM HEPES-KOH pH7.0 (Nacalai tesque), 150 mM Potasium acetate (KOAc; Nacalai tesque), 2 mM Magnesium acetate (MgOAc; Nacalai tesque), 2 μM Leupeptin (Peptide institute), 20% (W/V) Glycerol (Nacalai tesque), 50 μg/ml Bentonite (Nacalai tesque)] を, 1.5 ml 加え, 氷冷下でペッスルを5回上下させた。 これを, 20,000×g, 4°C, 30分遠心し, その上清を丁寧に回収した。上清に組織片が浮遊している場合は，さらに，20,000×g, 4°C,15分遠心した。 これを以降，翻訳系Lysateと呼ぶ。
3-2. Preparation of Organ Extract The frozen posterior silk glands were transferred to an ice-cooled 0.75% NaCl solution and thawed gently. After complete thawing is confirmed, excess water adhering to the organs is removed with filter paper, and this is pre-cooled with ice-cooled Loose type glass downs-type homogenizer (diameter 13 mm, length 82 mm, cylinder length 175 mm). , Wheaton). Ice-cooled extraction buffer for 1.5 g of posterior silk gland [20 mM HEPES-KOH pH7.0 (Nacalai tesque), 150 mM Potasium acetate (KOAc; Nacalai tesque), 2 mM Magnesium acetate (MgOAc; Nacalai tesque), 2 μM Leupeptin (Peptide institute), 20% (W / V) Glycerol (Nacalai tesque), 50 μg / ml Bentonite (Nacalai tesque)] was added in an amount of 1.5 ml, and the pestle was moved up and down 5 times under ice cooling. This was centrifuged at 20,000 × g, 4 ° C for 30 minutes, and the supernatant was carefully collected. If tissue fragments were suspended in the supernatant, they were further centrifuged at 20,000 xg, 4 ° C, for 15 minutes. This is hereafter referred to as the translation system Lysate.
3-3. 反応
無細胞翻訳反応液 [20 mM HEPES-KOH, pH 7.0, 125 mM KOAc, 1.75 mM MgOAc, 0.75 mM ATP, 1 mM GTP, 25 mM Creatin phosphate (CP), 200 μg/ml Creatin kinase (CK), 6% (w/v) Glycerol, 1 mM DTT, 40 μM Amino Acid Mixture, 250 μM EGTA, 1.5 U/μl RNase Inhibitor, 100 μg/ml tRNA, 160 μg/ml 前項において調製したさまざま構造をもつルシフェラーゼmRNA, 7.5 μl Lysate] は総量25 μlとして25°C下で反応させた。結果を図1及び図2に示す。図1は，ルシフェラーゼの発光量から測定した相対量を，図2はそれをウエスタンブロットで検討した結果である。
3-3. Reaction Cell-free translation reaction solution [20 mM HEPES-KOH, pH 7.0, 125 mM KOAc, 1.75 mM MgOAc, 0.75 mM ATP, 1 mM GTP, 25 mM Creatin phosphate (CP), 200 μg / ml Creatin kinase (CK), 6% (w / v) Glycerol, 1 mM DTT, 40 μM Amino Acid Mixture, 250 μM EGTA, 1.5 U / μl RNase Inhibitor, 100 μg / ml tRNA, 160 μg / ml Various structures prepared in the previous section Luciferase mRNA, 7.5 μl Lysate] was reacted at 25 ° C with a total volume of 25 μl. The results are shown in FIGS. 1 and 2. Fig. 1 shows the relative amount measured from the luminescence amount of luciferase, and Fig. 2 shows the result of examining it by Western blotting.
4. 無細胞転写-翻訳共役反応系
4-1. 器官抽出液の作製
凍結状態の後部絹糸腺を, 氷冷した0.75% NaCl 溶液に移し, 穏やかに解凍した。完全に解凍が確認された後, 器官に付着する余分な水分をろ紙で取り除き, これを, あらかじめ氷冷したLoose タイプのガラス製ダウンス型ホモジナイザー （直径 13 mm, 長さ 82 mm, 筒長175 mm, Wheaton）に移した。後部絹糸腺 1.5 g に対して氷冷した抽出緩衝液[10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-KOH pH 7.8 (Nacalai tesque), 2 μM Leupeptin (ペプチド研究所), 20% (W/V) Glycerol (Nacalai tesque), 50 μg/ml Bentonite (Nacalai tesque)] を, 1.5 ml 加え, 氷冷下でペッスルを4回上下させた。これを, 20,000×g, 4°C, 30分遠心し, その上清を丁寧に回収した。上清に組織片が浮遊している場合は，さらに，20,000×g, 4°C,15分遠心した。これを以降，ライセート (Lysate) と呼ぶ。
4. Cell-free transcription-translation conjugated reaction system
4-1. Preparation of Organ Extract The frozen posterior silk glands were transferred to an ice-cooled 0.75% NaCl solution and thawed gently. After complete thawing is confirmed, excess water adhering to the organs is removed with filter paper, and this is pre-cooled with ice-cooled Loose type glass downs-type homogenizer (diameter 13 mm, length 82 mm, cylinder length 175 mm). , Wheaton). Ice-cooled extraction buffer for 1.5 g of posterior silk gland [10 mM HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid)-KOH pH 7.8 (Nacalai tesque), 2 μM Leupeptin (Peptide Institute), 20% (W / V) Glycerol (Nacalai tesque), 50 μg / ml Bentonite (Nacalai tesque)] was added in an amount of 1.5 ml, and the pestle was moved up and down four times under ice cooling. This was centrifuged at 20,000 × g, 4 ° C for 30 minutes, and the supernatant was carefully collected. If tissue fragments were suspended in the supernatant, they were further centrifuged at 20,000 xg, 4 ° C, for 15 minutes. This is hereafter referred to as Lysate.
4-2. 反応
反応系は [2 M potassium acetate (KOAc; Nacalai tesque) 1.4 μl , 100 mM magnesium acetate (MgOAc ; Nacalai tesque) 0.4 μl , 10 mM adenosine triphosphate (ATP; Promega) 1.0 μl, 10 mM uridine triphosphate (UTP; Promega) 1.0 μl, 10 mM cytidine triphosphate (CTP; Promega) 1.0 μl, 10 mM guanosine triphosphate (GTP; Promega) 1.0 μl, 2.5 mM dithiothreitol (DTT) 0.5 μl , 10 mM spermidine 0.5 μl , 19 U/μl T7 RNA polymerase (Promega) 2.0 μl, Lysate 2.5 μl] に，第2-2.項で精製したプラスミドを適当量添加し，これに精製水を加えることで，反応液25 μl とした。反応は28°Cで行った。結果を図3〜図6に示す。図3は，ルシフェラーゼの発光量から測定した相対量を，図4はそれをウエスタンブロットで検討した結果である。また，配列の特定化（deletion）プラスミドを使っての，転写・翻訳共役型の合成の結果を図5，図6に示す。図5は，ルシフェラーゼの発光量から測定した相対量を，図6はそれをウエスタンブロットで検討した結果である。
4-2. Reaction The reaction system is [2 M potassium acetate (KOAc; Nacalai tesque) 1.4 μl, 100 mM magnesium acetate (MgOAc; Nacalai tesque) 0.4 μl, 10 mM adenosine triphosphate (ATP; Promega) 1.0 μl, 10 mM uridine. triphosphate (UTP; Promega) 1.0 μl, 10 mM cytidine triphosphate (CTP; Promega) 1.0 μl, 10 mM guanosine triphosphate (GTP; Promega) 1.0 μl, 2.5 mM dithiothreitol (DTT) 0.5 μl, 10 mM spermidine 0.5 μl, 19 U To / μl T7 RNA polymerase (Promega) 2.0 μl, Lysate 2.5 μl], an appropriate amount of the plasmid purified in Section 2-2. Was added, and purified water was added to make a reaction solution of 25 μl. The reaction was carried out at 28 ° C. The results are shown in FIGS. 3 to 6. Fig. 3 shows the relative amount measured from the luminescence amount of luciferase, and Fig. 4 shows the result of examining it by Western blotting. In addition, Fig. 5 and Fig. 6 show the results of transcription / translation-conjugated synthesis using a sequence-specification plasmid. Fig. 5 shows the relative amount measured from the luminescence amount of luciferase, and Fig. 6 shows the result of examining it by Western blotting.
5. ルシフェラーゼの定量
ルシフェラーゼ活性の測定は，反応系から2.5 μl を取り出し，あらかじめ28℃に保温したLuciferase Assay System (Promega) 50 μl と混合し，これを速やかに96穴タイタープレート (Nalge Nunc) に移して，ルミノメーター(Luminescencer-JNRAB2100; ATTO) で発光量を測定した．測定は25℃下で，Delay 3 秒，Integrate 5 秒の計測条件でおこない，発光積算量は機械に付属するソフトで処理することにより，求めた。
5. Quantification of luciferase To measure luciferase activity, take 2.5 μl from the reaction system, mix it with 50 μl of Luciferase Assay System (Promega) preheated to 28 ° C, and immediately put it on a 96-well titer plate (Nalge Nunc). After transfer, the amount of luminescence was measured with a luminometer (Luminescencer-JNRAB2100; ATTO). The measurement was performed at 25 ° C under the measurement conditions of
得られた発光積算量からは，あらかじめQuantium Reconbinant Luciferase (Promega) を用いて作成した検量線を元に，ルシフェラーゼタンパク質量を決定した。 From the obtained integrated luminescence, the amount of luciferase protein was determined based on the calibration curve prepared in advance using Quantium Reconbinant Luciferase (Promega).
6. Western blot解析
Western blotting法は，Towbin et al. (1979) とKyhse and Audenseu (1984) の方法に従った。
6. Western blot analysis
Western blotting followed the method of Towbin et al. (1979) and Kyhse and Audenseu (1984).
6-1. サンプルの調製
反応開始5時間後の翻訳反応液および，転写‐翻訳反応液から取り出した溶液10 μlに対して，サンプル緩衝液 [4% SDS，5% Mercaptoethanol，10% Glycerol，0.002% Bromophenol blue，125mM Tris-HCl (pH 6.8) ] を等量加え，100℃で5分間処理した。
6-1. Sample preparation For 10 μl of the
6-2. 電気泳動(SDS-polyacrylamide gel electrophoresis;SDS-PAGE)
電気泳動はLaemmli (1970) の方法に従った。8×10，厚さ1 mmのミニスラブゲルに12.5% 分離ゲル [30% (w/v) Acrylamide-0.8% (w/v) Bisacrylamide混合液4 ml，1.5M Tris-HCl (pH8.8) 緩衝液 2.5 ml，10% SDS溶液 100 μl，H2O 3 ml，N,N,N’,N’,- Tetrametylethylenediamine (TEMED) 10 μl，10% Ammonium peroxodisulfate (APS) 溶液 100 μlを混合した] を流し込み，ゲルが固まるまで放置した。分離ゲルが固化した後，濃縮ゲル溶液 [30% (w/v) Acrylamide-0.8% (w/v) Bisacrylamide混合液0.79 ml，0.5 M Tris-HCl (pH 6.8) 緩衝液 1.25 ml，10% SDS溶液 50 μl，H2O 2.8 ml，TEMED 10 μl，10% APS溶液 50 μlを混合した] をさらに流し込んだ。各レーンには，第8-1項で調整したサンプルをシリンジで 10 μlずつ添加した後，泳動緩衝液 (0.025M Tris，0.192M Glycine，0.1% SDS)を用いて室温，150 V 定電圧で泳動した。
6-2. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Electrophoresis followed the method of Laemmli (1970). 12.5% Separation Gel [30% (w / v) Acrylamide-0.8% (w / v) Bisjapan Mixture 4 ml, 1.5 M Tris-HCl (pH 8.8) Buffer) on 8 x 10 1 mm thick mini slab gel Solution 2.5 ml, 10
6-3. ポリアクリルアミドゲルから膜への転写法
SDS-PAGEによって分離されたタンパク質は，Polyvinilden difluoride膜 (PVDF膜) を用いて電気的に転写した。PVDF膜の陽極側の電極に，分離ゲルの大きさに合わせて切断したろ紙を転写用緩衝液 (20mM Glycine, 25mM Tris, 20% Methanol) に浸したものを3枚置き，その上にMethanolで1分振盪した後転写用緩衝液に浸したPVDF膜，次にポリアクリルアミドゲルを重ね，さらにその上に前述の転写用緩衝液を染み込ませたろ紙を3枚重ね，100mA 定電流，室温で1時間通電した。
6-3. Transfer method from polyacrylamide gel to membrane
Proteins separated by SDS-PAGE were electrically transcribed using a Polyvinilden difluoride membrane (PVDF membrane). On the electrode on the anode side of the PVDF film, place three sheets of filter paper cut according to the size of the separation gel soaked in transfer buffer (20 mM Glycine, 25 mM Tris, 20% Methanol), and use Methanol on it. After shaking for 1 minute, a PVDF film soaked in a transfer buffer solution, then a polyacrylamide gel is layered, and then 3 sheets of filter paper impregnated with the above-mentioned transfer buffer solution are layered on top of each other. Energized for hours.
6-4. 免疫学的検出法
転写後のPVDF膜をD.W.で三回洗浄した後，ブロッキング溶液 (0.1% スキムミルクを含むT-PBS[137 mM NaCl，2.7 mM KCl，8.1 mM NaHPO4・12H2O，1.5 mM KH2PO，0.1% Polyoxyetylene Sorbitan (Tween 20)] ) 中で2時間，室温で振盪した。反応後，PVDF膜をT-PBSで1分間，室温で振盪し，この操作を3回繰り返した。一次抗体として，Anti-Luciferase pAb (Promega) をT-PBSで1,000倍希釈したものを用い，1時間，室温で振盪した後，さらに一晩，4℃でPVDF膜を反応させた。反応後，PVDF膜はT-PBSで10分間，室温で振盪し，この操作を3回繰り返した。二次抗体として，Peroxidase-conjugated Donkey Anti-Goat IgG (Promega) をT-PBSで50,000倍希釈したものを用い，1時間，室温で振盪した。反応後，PVDF膜をT-PBSで10分間，室温で振盪し，この操作を3回繰り返した。ブロットの発色はイムノスターLD (和光純薬工業) を用い，キットに添付された方法に従い，X線フィルムに感光させて，可視化した。
6-4. After the PVDF membrane after immunological detection methods transcription was washed three times with DW, blocking solution (T-PBS [137 mM NaCl containing 0.1% skim milk, 2.7 mM KCl, 8.1 mM NaHPO 4 · 12H 2 It was shaken in O, 1.5 mM KH 2 PO, 0.1% Polyoxyetylene Sorbitan (Tween 20)]) for 2 hours at room temperature. After the reaction, the PVDF membrane was shaken with T-PBS for 1 minute at room temperature, and this operation was repeated 3 times. Anti-Luciferase pAb (Promega) diluted 1,000-fold with T-PBS was used as the primary antibody, shaken at room temperature for 1 hour, and then reacted with a PVDF membrane at 4 ° C. overnight. After the reaction, the PVDF membrane was shaken with T-PBS for 10 minutes at room temperature, and this operation was repeated 3 times. As a secondary antibody, Peroxidase-conjugated Donkey Anti-Goat IgG (Promega) diluted 50,000 times with T-PBS was used and shaken at room temperature for 1 hour. After the reaction, the PVDF membrane was shaken with T-PBS for 10 minutes at room temperature, and this operation was repeated 3 times. The color of the blot was visualized by using Immunostar LD (Wako Pure Chemical Industries, Ltd.) and exposing it to an X-ray film according to the method attached to the kit.
なお、ABPVの汎用優位性については，TnT T7 Insect Cell Extract Protein Expression System (Promega) -昆虫細胞Sf21エクストラクトで確認している。 The general-purpose superiority of ABPV has been confirmed with TnT T7 Insect Cell Extract Protein Expression System (Promega) -Insect Cell Sf21 Extract.
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