Bac-to-Bac™ Baculovirus Expression System

Citations & References (66)

Gibco™

Bac-to-Bac™ Baculovirus Expression System

Bac-to-Bac Baculovirus Expression Systemを使用すると、昆虫細胞の発現試験で組換えバキュロウイルスを効率的に生成できます。このシステムは昆虫細胞の相同組換えではなく、大腸菌における部位に特異的な転位による組換えバキュロウイルスの生成に依存します。このシステムの特長は以下のとおりです。•時間を節約する発現バクミド— Bac-to-Bacシステムでは詳細を見る

製品番号（カタログ番号）	数量
10359016	1 kit

製品番号（カタログ番号） 10359016

価格（JPY）

180,500

お問い合わせください ›

1 kit

Bac-to-Bac Baculovirus Expression Systemを使用すると、昆虫細胞の発現試験で組換えバキュロウイルスを効率的に生成できます。このシステムは昆虫細胞の相同組換えではなく、大腸菌における部位に特異的な転位による組換えバキュロウイルスの生成に依存します。このシステムの特長は以下のとおりです。

•時間を節約する発現バクミド— Bac-to-Bacシステムでは、pFastBacベクターの発現カセットがDH10Bac大腸菌コンピテントセルの親バクミドで組換えを行い、発現バクミドを形成します。その後、組換えバキュロウイルス粒子の生成のために、バクミドを昆虫細胞にトランスフェクションします。

•簡単なコロニースクリーニング—DH10Bac大腸菌の親バクミドは、lacZa遺伝子のセグメントを含みます。lacZa遺伝子が発現カセットの転位時に破壊されることによって、組換えコロニーの容易な識別のために組換えの青白選択が可能になります。

• ExpiFectamine Sf Transfection試薬での高いトランスフェクション効率—Bac-to-Bac TOPO発現キットには、迅速で柔軟なプロトコルを使用した効率的なDNAトランスフェクションを実現する次世代のExpiFectamine Sf Transfection試薬が付属しています。この試薬の詳細をご覧ください›

• 柔軟性—Bac-to-Bacシステムには、クローニングを簡素化するための大規模な複数のクローニング部位（MCS）を含むpFastBacベクターが付属しています。追加の2つのベクターは個別に提供されます。ヒスチジンタグ付き組換えタンパク質の生成のためのpFastBac HTベクターと、P10およびポリヘドリンプロモーターを使用して2つのタンパク質を同時に発現するためのpFastBac Dualベクター。

• 高タンパク質発現—pFastBacベクターは、強力なポリヘドリンプロモーターを使用して、Sf9細胞、Sf21細胞、High Five細胞などの幅広い昆虫細胞株で高レベルの発現を実現します。

研究用にのみ使用できます。診断用には使用いただけません。

製品タイプバキュロウイルス発現システム

数量1 kit

ベクターpFastBac

クローニング法制限酵素／MCS

製品ラインBac-to-Bac

プロモーターポリヘドリン

タンパク質タグタグなし

Unit SizeEach

組成および保存条件

各Bac-to-Bac Baculovirus Expression Systemには、次のものが含まれます。
• pFastBac1ベクター（10 µg）、-20℃で保管してください
• pFastBac 1-Gusコントロールベクター（4 ng）、-20℃で保管してください
• MAX Efficiency DH10Bacケミカルコンピテント大腸菌（5 x 0.1 mLの1キット）、-80℃で保管してください
• ExpiFectamine SF Transfection試薬（1 mL）、4℃で保管してください。凍結されません

よくあるご質問（FAQ）

I cannot grow this white colony in liquid culture. What should I do?

The concentration of gentamicin might be too high. Try lowering the amount to 5 µg/mL and try adding more of the colony to the culture medium.

What has happened when I see blue colonies? How about colonies which are blue in the center and white on the edges?

In the case of a blue colony, the E. coli has the bacmid and the plasmid in it, allowing the cells to survive the selection process. However, because the transposition has not occurred, the LacZ gene is not disrupted. For bulls-eye colonies, this indicates that the transposition took place when the colony was growing. Re-streaking for an isolated clone from the white portion of the mixed colony should yield some colonies where transposition occurred.

I'm getting mostly white/wild-type plaques instead of blue/recombinant plaques. What am I doing wrong?

This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.

I've infected my cells and see large polyhedra in one cell and smaller polyhedra (more numerous) in a neighboring cell. Is this normal?

Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3-4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.

I'm worried that I am not getting plaques. How many days does it take to see plaques and what size are they typically?

Normally, very small white dots show up about 5-7 days and 1 mm plaques show up around day 10. Plaques can vary in size from 1 mm to 4 mm.

View all Bac-to-Bac™ Baculovirus Expression System FAQs

引用および参考文献 (66)

引用および参考文献

Abstract

The vomeronasal receptor V2R2 does not require escort molecules for expression in heterologous systems.

Authors:Silvotti L, Giannini G, Tirindelli R,

Journal:Chem Senses

PubMed ID:15647459

'In rodents, many behavioural responses are triggered by pheromones. These molecules are believed to bind and activate two families of G-protein coupled receptors, namely V1Rs and V2Rs, which are specifically expressed in the chemosensory neurons of the vomeronasal organ. V2Rs are homologous with Group 3 of G-protein-coupled receptors, which includes ... More

Identification of Novel SH3 Domain Ligands for the Src Family Kinase Hck. WISKOTT-ALDRICH SYNDROME PROTEIN (WASP), WASP-INTERACTING PROTEIN (WIP), AND ELMO1.

Authors: Scott Margaret Porter; Zappacosta Francesca; Kim Eun Young; Annan Roland S; Miller W Todd;

Journal:J Biol Chem

PubMed ID:12029088

'The importance of the SH3 domain of Hck in kinase regulation, substrate phosphorylation, and ligand binding has been established. However, few in vivo ligands are known for the SH3 domain of Hck. In this study, we used mass spectrometry to identify approximately 25 potential binding partners for the SH3 domain ... More

Allocation of helper T-cell epitope immunodominance according to three-dimensional structure in the human immunodeficiency virus type I envelope glycoprotein gp120.

Authors: Dai G; Steede N K; Landry S J;

Journal:J Biol Chem

PubMed ID:11551929

'The specificity and intensity of CD4(+) helper T-cell responses determine the effectiveness of immune effector functions. Promiscuously immunodominant helper T-cell epitopes in the human immunodeficiency virus (HIV) envelope glycoprotein gp120 could be important in the development of broadly protective immunity, but the underlying mechanisms of immunodominance and promiscuity remain poorly ... More

Expression, purification, and characterization of an activated cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase from baculovirus-infected insect cells.

Authors: Cai X Y; Shanahan M; Miller K; Gommoll C; Lundell D; Zavodny P; Dalie B;

Journal:Protein Expr Purif

PubMed ID:9226723

'An activated form of the human cytokine-suppressive anti-inflammatory drug-binding protein 2 (CSBP2) kinase was expressed in Spodoptera frugiperda (SF9) cells from a baculovirus vector. To maximize expression and to facilitate purification of the recombinant protein, CSBP2 kinase was expressed as a carboxy-terminal fusion protein to glutathione S-transferase (GST). Under optimal ... More

Kinesin Superfamily Motor Protein KIF17 and mLin-10 in NMDA Receptor-Containing Vesicle Transport

Authors:Mitsutoshi Setou, Terunaga Nakagawa, Dae-Hyun Seog, Nobutaka Hirokawa *

Journal:Science

PubMed ID:10846156

'Experiments with vesicles containing N-methyl-D-aspartate (NMDA)receptor 2B (NR2B subunit) show that they are transported alongmicrotubules by KIF17, a neuron-specific molecular motor in neuronaldendrites. Selective transport is accomplished by direct interaction of theKIF17 tail with a PDZ domain of mLin-10 (Mint1/X11), which is aconstituent of a large protein complex including mLin-2 ... More

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