Name: using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy
Uploaded: 2011-11-01T12:00:00
Description: Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

この作品は、グラントの数5エクセレンスの西部地域センター（WRCE）を通じて、U54 AI057156 - 07、アレルギーや国立感染症研究所から1 R01 AI08764301 - A1、および大学のワクチン開発のためのシーリーセンターの内部資金によって賄われていたテキサスメディカルブランチ。

1。プラスミドDNAから組換えMP - 12エンコーディングのNSSの変異（S）2の回収<ol><li>スプレッドベビーハムスター腎臓（BHK）最小必須培地（MEM）-α（Invitrogen社、カタログ番号32561037）を含む10％ウシ胎児血清（FBS 6 cmのディッシュに安定的に発現するT7 RNAポリメラーゼ/ T7 - 9細胞15、、 ）、ペニシリン - ストレプトマイシン（ペニシリン：100 U / mlと、ストレプトマイシン：100μg/ ...

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RVFVのために遺伝学のシステムを逆には、T7プロモーター2,4,5またはマウス3またはヒトの4 POL - Iプロモーターを利用していくつかのグループによって開発されている。この原稿では、我々はBHK/T7-9セル15その安定的に発現するT7 RNAポリメラーゼを用いて組換えRVFV MP - 12株を生成するためのプロトコルについて説明します。 BHK/T7-9細胞の状態によって異なるウイル...

<table border="1"><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（省略可能） </td></tr><tr><td>最小必須培地（MEM）、アルファ</td><td>インビトロジェン</td><td> 32561037 </td><td></td></tr><tr><td>ダルベッコ改変最小必須培地</td><td>インビトロジェン</td><td> 11965092 </td><td></td></tr><tr><td>改変イーグル培地（MEM 2倍） </td><td>インビトロジェン</td><td> 11935046 </td><td></td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>インビトロジェン</td><td> 15140122 </td><td></td></tr><tr><td>ハイグロマイシンB </td><td> Cellgro </td><td> 30から240 - CR </td><td></td></tr><tr><td>トリプトースリン酸塩スープ</td><td> MPのバイオメディカル</td><td> 1682149 </td><td></td></tr><tr><td> Noble寒天</td><td> VWR </td><td> 101170-362 </td><td></td></tr><tr><td>トランジット- LT1 </td><td> Mirus </td><td> MIR2300</td><td></td></tr><tr><td>のOpti - MEM </td><td>インビトロジェン</td><td> 31985070 </td><td></td></tr><tr><td>エアロゾルタイトな蓋</td><td>エッペンドルフ</td><td> C - 2223〜25 </td><td></td></tr><tr><td> 0.33パーセントニュートラルレッド溶液</td><td>シグマアルドリッチ</td><td> N2889 - 100ML </td><td></td></tr><tr><td> C57/WT MEF細胞</td><td> InvivoGen </td><td> MEF - c57wt </td><td></td></tr><tr><td>ブラストサイジンS </td><td> InvivoGen </td><td> ANT - BL - 1 </td><td></td></tr><tr><td>ゼオシン</td><td> InvivoGen </td><td>アリ - 亜鉛- 1 </td><td></td></tr><tr><td>定量ブルー</td><td> InvivoGen </td><td> REP - QB1 </td><td></td></tr><tr><td> BHK/T7-9細胞15 </td><td>岐阜大学、日本</td><td></td><td></td></tr><tr><td>ベロE6細胞</td><td> ATCC </td><td> CRL - 1586 </td><td></td></tr></table>

using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy

出血熱、神経疾患やヒトの失明、そして高レートの中絶と反芻動物1の胎児の奇形を引き起こすリフトバレー熱ウイルス（RVFV）は、選択剤とリスクグループ3の病原体を重ねるHHS / USDAとして分類されている。それは、ブニヤウイルス科の属Phlebovirusに属し、このファミリーの中で最も毒性の強いメンバーの一人です。 RVFV MP - 12ワクチン株2,3だけでなく、ZH548とZH501を含む野生型RVFV株4-6、、のためのいくつかのリバースジェネティクスシステムが2006年から開発されている。 MP - 12株は、（リスクグループ2病原体および非選択剤である）のM -およびL -セグメントでいくつかの変異によって高度に弱毒化ですが、それでも機能的な病原性をコード病原性S -セグメントRNA 3を 、運ぶ要因、NSS。それはとしてeffic複製しながらNSS ORFの欠失、フレームの69％を運ぶrMP12 - C13type（C13type）は、すべての既知のNSSの機能を欠いているIENTは、VeroE6細胞におけるMP - 12は、タイプ- I IFNを欠いていません。 NSSは、インターフェロン（IFN）-βmRNAの7,8を含むホストの転写のシャットオフを誘導し、翻訳後レベルでの二本鎖RNA依存性プロテインキナーゼ（PKR）の分解を促進する。9,10 IFN -βの転写であるインターフェロン調節因子3（IRF - 3）、NF - kBからアクチベーター蛋白質1（AP - 1）、およびIFN-alpha/beta受容体へのIFN -βの結合（IFNAR）によって上方制御は、IFN -αの転写を刺激するNSSによるIFN -β遺伝子を含むホストの転写抑制はウイルス複製に応答してそれらのISGsの遺伝子のupregulationsを防ぐ一方で、宿主の抗ウイルス活性を誘導する遺伝子または他のインターフェロン刺激遺伝子（ISGs）11、もののIRF - 3、NF - kBから活性化因子タンパク質- 1（AP - 1）RVFV7によって活性化することができます。 。このように、NSSはさらにMP - 12を減衰させる、およびIFN -β抑制機能を廃止することによって宿主自然免疫応答を高めるために優秀なターゲットです。ここ、我々は組換えMP - 12エンコーディング変異NSSを生成するためのプロトコルを記述し、IFN -βmRNAの合成を抑制する機能を欠いているNSS変異体を同定するためのスクリーニング方法の例を提供しています。自然免疫におけるその重要な役割に加えて、I型IFNは、樹状細胞と適応免疫応答12-14誘導の成熟にとって重要です。従って、I型IFNを誘導するNSSの変異体は、さらに減衰されますが、同時にそれらのワクチン接種のアプローチのための理想的な候補になる野生型MP - 12、より宿主の免疫応答を刺激するより効率的です。

Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

リフトバレー熱ウイルスMP - 12ワクチン株の逆遺伝学的システムは、追加のMP - 12の増加減衰および免疫原性と変異体を作成するための便利なツールです。我々はNSS変異株を生成し、特徴付けるためにプロトコルについて説明します。

リフトバレー熱ウイルスワクチンの安全性および有効性を改善するためのMP - 12株のNSS遺伝子を操作する逆遺伝学を使用して

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In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

遺伝子または薬物療法で使用するためのHIV-1産生を標的RNAの有効性および毒性の評価

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

エスケープ中和抗体でインフルエンザ ウイルスの亜種の世代

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

ヒトの過剰発現と精製<em&gt;シス</em&gt; - プレニルトランスフェラーゼ<em&gt;大腸菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

系統解析を使用して真核生物遺伝子の起源を調査するには

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Assembly and Purification of Prototype Foamy Virus Intasomes

アセンブリおよびプロトタイプ フォーミー ウイルス Intasomes の精製

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主-病原体応答とマウスでワクチンの有効性の評価

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...