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

Questo lavoro è stato finanziato per numero di Grant 5 U54 AI057156-07 tramite Western Centro Regionale di Eccellenza (WRCE), 1 AI08764301 R01-A1 da Istituto Nazionale di Allergologia e Malattie Infettive, e un finanziamento interno da Sealy Centro per lo sviluppo di vaccini presso l&#39;Università di Texas Medical Branch.

1. Recupero di mutazione ricombinante-12 MP codifica NSS (s) da plasmide DNA 2 <ol><li> Diffusione renali di criceto neonato (BHK) / T7-9 celle 15, che esprimono stabilmente T7 RNA polimerasi, in 6 cm di piatti in Medium minimo (MEM)-alfa (Invitrogen, Cat # 32561037) contenente il 10% siero fetale bovino (FBS ), penicillina-streptomicina (Penicillina: 100 U / ml, streptomicina: 100 mcg / ml) (Invitrogen, Cat # 15140122), e 600 mg / ml di Hygromycin B (Cellgro, Cat # 30-240-CR). * L&#39;eff...

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Sistemi di genetica inversa per RVFV sono stati sviluppati da diversi gruppi utilizzando promotore T7 2,4,5 o mouse 3 o umano 4 pol-I promoter. In questo manoscritto, si descrive un protocollo per generare ricombinante RVFV MP-12 ceppi utilizzando BHK/T7-9 15 cellule che esprimono stabilmente T7 RNA polimerasi. L&#39;efficienza di recupero virale varia a seconda delle condizioni di BHK/T7-9 cellule, la quantità di plasmidi, il numero di cellule transfettate e così via. Abbia...

<table border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Medium minimo (MEM)-alfa </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Dulbecco modificato medio minimo indispensabile </td><td> Invitrogen </td><td> 11965092 </td><td></td></tr><tr><td> Modified Eagle Medium (MEM 2x) </td><td> Invitrogen </td><td> 11935046 </td><td></td></tr><tr><td> Penicillina-Streptomicina </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> Triptosio fosfato brodo </td><td> MP Biomedicals </td><td> 1682149 </td><td></td></tr><tr><td> Agar Noble </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 coperchio stretto </td><td> Eppendorf </td><td> C-2223-25 </td><td></td></tr><tr><td> 0,33% soluzione di rosso neutro </td><td> Sigma Aldrich </td><td> N2889-100ML </td><td></td></tr><tr><td> C57/WT cellule MEF </td><td> InvivoGen </td><td> mef-c57wt </td><td></td></tr><tr><td> Blasticidin S </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-blu </td><td> InvivoGen </td><td> rep-QB1 </td><td></td></tr><tr><td> BHK/T7-9 celle 15 </td><td> Gifu University, in Giappone </td><td></td><td></td></tr><tr><td> Vero E6 cellule </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

Virus della febbre della Rift Valley (RVFV), che causa febbre emorragica, disturbi neurologici o cecità negli esseri umani, e un alto tasso di aborti e malformazioni fetali nei ruminanti 1, è stata classificata come HHS / USDA sovrapposizione agente di selezionare e di un gruppo di rischio 3 patogeno. Appartiene al genere Phlebovirus nel Bunyaviridae famiglia ed è uno dei membri più virulenta di questa famiglia. Diversi sistemi di genetica inversa per l&#39;MP-12 RVFV ceppo del vaccino 2,3 così come wild-type ceppi RVFV 4-6, tra cui ZH548 e ZH501, sono state sviluppate dal 2006. La MP-12 ceppo (che è un gruppo di rischio 2 patogeno e un non-selezione agente) è fortemente attenuata da diverse mutazioni nel suo M-e L-segmenti, ma porta ancora virulento S-segmento RNA 3, che codifica per una virulenza funzionale fattore, NSS. Il rMP12-C13type (C13type) portando al 69% in-frame cancellazione di NSS ORF manca di tutte le funzioni NSS noto, mentre si replica come efficient così come MP-12 in VeroE6 cellule prive di tipo I IFN. NSS induce una chiusura di trascrizione ospitare tra cui l&#39;interferone (IFN)-beta mRNA 7,8 e promuove la degradazione della doppia elica RNA-dipendente proteina chinasi (PKR) al post-traduzionali livello. 9,10 IFN-beta è trascrizionalmente sovraregolati per il fattore normativo interferone 3 (IRF-3), NF-kB e proteina attivatrice-1 (AP-1), e il legame di IFN-beta di IFN-alpha/beta recettore (IFNAR) stimola la trascrizione di IFN-alfa geni o altri geni dell&#39;interferone stimolata (ISGs) 11, che induce ospitare attività antivirale, mentre la trascrizione soppressione ospiti ivi compresi IFN-beta gene NSS impedisce la upregulations gene di quelli ISGs in risposta alla replicazione virale nonostante IRF-3, NF-kB e attivatore proteina-1 (AP-1) può essere attivata RVFV7. . Così, NSS è un obiettivo eccellente per attenuare ulteriormente MP-12, e per migliorare la risposta immunitaria innata ospitare abolendo l&#39;IFN-beta funzione di soppressione. Qui, Si descrive un protocollo per la generazione di un ricombinante MP-12 NSS codifica mutato, e fornire un esempio di un metodo di screening per identificare i mutanti NSS manca la funzione di sopprimere IFN-beta sintesi di mRNA. Oltre al suo ruolo essenziale nella dell&#39;immunità innata, tipo I IFN è importante per la maturazione delle cellule dendritiche e l&#39;induzione di una risposta immunitaria adattativa 12-14. Così, mutanti NSS indurre IFN di tipo I sono ulteriormente attenuati, ma allo stesso tempo sono più efficienti a stimolare le risposte immunitarie di ospitare wild-type MP-12, che li rende candidati ideali per gli approcci di vaccinazione.

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

Il sistema di genetica inversa per la Valle del virus della febbre della Rift MP-12 ceppo vaccinale è un utile strumento per la creazione di ulteriori MP-12 mutanti con attenuazione aumentata e l&#39;immunogenicità. Noi descriviamo il protocollo per generare e caratterizzare NSS ceppi mutanti.

Utilizzando genetica inversa per manipolare il gene del NSS il virus della febbre della Rift Valley MP-12 Strain per migliorare la sicurezza e l&#39;efficacia del vaccino

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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Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Valutazione delle risposte ospite-patogeno e l'efficacia del vaccino nei topi

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