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

Bu çalışma, Hibe sayısı 5 Mükemmellik Batı Bölge Merkezi (WRCE) yoluyla U54 AI057156-07, 1 R01 AI08764301-A1 Ulusal Alerji ve Bulaşıcı Hastalıklar Enstitüsü ve Üniversitesi&#39;nde bir iç finansman Sealy Aşı Geliştirme Merkezi tarafından finanse edildi Texas Medical Branch.

1. Plazmid DNA&#39;lar 2 rekombinant MP-12 kodlama NSS mutasyon (lar) Kurtarma <ol><li> Spread bebek bir hamster böbrek (BHK) / T7-9 hücreler 15, stabil bir şekilde ifade T7 RNA polimeraz, 6 cm kaplarına Minimum Essential Medium (MEM)-alfa (Invitrogen, Kedi # 32.561.037% 10 fetal sığır serumu (FBS içeren) ), Penisilin-Streptomisin (Penisilin: 100 U / ml, Streptomisin: 100 mg / ml) (Invitrogen, Kedi # 15.140.122) ve 600 mcg / ml hygromycin B (Cellgro, Cat # 30-240-CR). * Viral iyile...

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RVFV için genetik sistemleri Ters T7 organizatörü 2,4,5 veya 3 fare veya insan 4 pol-I organizatörü kullanılarak çeşitli gruplar tarafından geliştirilmiştir. Bu yazıda, rekombinant RVFV MP-12 suşları, 15 stabil bir şekilde ifade T7 RNA polimeraz BHK/T7-9 hücreleri kullanarak oluşturmak için bir protokol açıklar. BHK/T7-9 hücrelerin durumuna göre farklılıklar viral geri kazanım verimliliği, plazmid miktarı, transfekte hücreleri ve benzeri numarası. ...

<table border="1"><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Minimum Essential Medium (MEM)-alfa </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Dulbecco&#39;nun değiştirilmiş asgari temel orta </td><td> Invitrogen </td><td> 11965092 </td><td></td></tr><tr><td> Modifiye Kartal Orta (MEM 2x) </td><td> Invitrogen </td><td> 11935046 </td><td></td></tr><tr><td> Penisilin-Streptomisin </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> Hygromycin B </td><td> Cellgro </td><td> 30-240-CR </td><td></td></tr><tr><td> Tryptose fosfat broth </td><td> MP Biomedicals </td><td> 1682149 </td><td></td></tr><tr><td> Noble Agar </td><td> VWR </td><td> 101170-362 </td><td></td></tr><tr><td> Transit LT1 </td><td> Mirus </td><td> MIR2300</td><td></td></tr><tr><td> Opti-MEM </td><td> Invitrogen </td><td> 31985070 </td><td></td></tr><tr><td> Aerosol sıkı kapak </td><td> Eppendorf </td><td> C-2223-25 </td><td></td></tr><tr><td> % 0.33 nötral kırmızı çözüm </td><td> Sigma Aldrich </td><td> N2889-100ML </td><td></td></tr><tr><td> C57/WT MEF hücreleri </td><td> InvivoGen </td><td> MEF-c57wt </td><td></td></tr><tr><td> Blasticidin G </td><td> InvivoGen </td><td> Ant-bl-1 </td><td></td></tr><tr><td> Zeocin </td><td> InvivoGen </td><td> ant-zn-1 </td><td></td></tr><tr><td> Quanti-Mavi </td><td> InvivoGen </td><td> rep-qb1 </td><td></td></tr><tr><td> BHK/T7-9 hücreler 15 </td><td> Gifu Üniversitesi, Japonya </td><td></td><td></td></tr><tr><td> Vero E6 hücreleri </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

Hemorajik ateş, nörolojik bozukluklar veya insanlarda körlüğe ve yüksek oranda kürtaj ve geviş getiren hayvanların 1 fetal malformasyon neden Rift Vadisi ateşi virüsü (RVFV), bir HHS / USDA seçin ajan ve 3 patojen bir risk grubu örtüşüyor olarak sınıflandırılmıştır. Bu, aile Bunyaviridae cinsi Phlebovirus ait ve bu ailenin en şiddetli üyelerinden biridir . 2006 yılından bu yana RVFV MP-12 aşı suşu 2,3 ZH548 ve ZH501 dahil olmak üzere yabani tip RVFV suşları 4-6, yanı sıra pek çok ters genetik sistemler geliştirilmiştir . MP-12 suşu (risk grubunda 2 patojen ve non-select ajan olan) birkaç kendi mutasyonlar M ve L-segmentleri oldukça zayıflatılmış, ancak hala fonksiyonel bir virülans kodlar öldürücü G-segment RNA 3 taşır faktörü, NSS. Effic çoğaltır rMP12 C13type (C13type) NSS ORF silme-kare% 69 taşıma, bilinen tüm NSS fonksiyonları yoksunient VeroE6 hücrelerinde MP-12 tip I IFN eksik yok. NSS, interferon (IFN)-beta mRNA 7,8 dahil olmak üzere, ev sahibi transkripsiyon kapatma indükler ve post-translasyon seviyesinde çift sarmallı RNA-bağımlı protein kinaz (PKR) bozulması teşvik 9,10 IFN-beta transkripsiyonel IFN-alfa interferon düzenleyici faktör 3 (IRF-3), NF-kB ve aktivatör protein-1 (AP-1), IFN-beta ve IFN-alpha/beta reseptör bağlayıcı (IFNAR) upregüle transkripsiyonu uyarır NSS IFN-beta gen dahil olmak üzere ana transkripsiyon bastırma viral replikasyon yanıt, bu ISGs gen upregulations engeller ise, ev sahibi antiviral aktivite gösteren genler veya diğer interferon uyarılan genlerin (ISGs) 11, rağmen IRF-3, NF-kB ve aktivatör protein-1 (AP-1) RVFV7 tarafından aktif hale getirilebilir. . Böylece, NSS, daha da azaltmak için MP-12 ve IFN-beta bastırma fonksiyonu kaldırılarak, doğuştan gelen bağışıklık yanıtlarını barındıran geliştirmek için mükemmel bir hedef. Burada, Rekombinant bir MP-12 kodlaması mutasyona uğramış NSS üretmek için bir protokol tanımlayan ve IFN-beta mRNA sentezini bastırmak için fonksiyonu olmayan NSS mutantlar tanımlamak için bir tarama yöntemi bir örnek oluşturmaktadır. Doğuştan gelen bağışıklık önemli rol ek olarak, tip I IFN, dendritik hücreler ve adaptif immün yanıt 12-14 indüksiyon olgunlaşması için çok önemlidir . Böylece, NSS mutantlar tip I IFN inducing daha da zayıflatılmış, ama aynı zamanda onları aşılama yaklaşımlar için ideal bir aday yapar yabani tip MP-12, daha teşvik edici bir konak bağışıklık yanıtlarını daha verimlidir.

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

Rift Vadisi ateşi virüs MP-12 aşı suşu için ters genetik sistemi ek MP-12 mutantlar artan zayıflama ve immünojenite oluşturmak için yararlı bir araçtır. Biz NSS mutant suşlar oluşturmak ve karakterize etmek için protokol açıklar.

Rift Vadisi Ateşi Virüsü Aşı Güvenliği ve Etkinliği Artırmak İçin MP-12 Gerinim NSS Gen işleyin Ters Genetik kullanma

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

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Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Aşırı Ezme ve İnsanın Saflaştırılması<em&gt; Cis</em&gt; -prenıltransferaz in<em&gt; Escherichia coli</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

Ökaryotik gen kökenli araştırmaya filogenetik analizi kullanılarak

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

Derleme ve prototip köpüklü virüs Intasomes arıtma

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

Ana bilgisayar-patojen yanıt ve aşı etkinliği farelerde değerlendirilmesi

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