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

Ce travail a été financé par la concession numéro 5 U54 AI057156-07 par le centre régional de l&#39;Ouest d&#39;excellence (WRCE), 1 R01-A1 AI08764301 de l&#39;Institut national des allergies et des maladies infectieuses, et d&#39;un financement interne du Centre de développement des vaccins Sealy à l&#39;Université de Texas Medical Branch.

1. Récupération des MP-12 recombinante mutation NSs encodage (s) à partir du plasmide ADN 2 <ol><li> Fais de rein de hamster bébé (BHK) / T7-9 cellules 15, qui expriment de façon stable la T7 RNA polymérase, en 6 cm de plats dans un milieu essentiel minimum (MEM)-alpha (Invitrogen, Cat # 32561037) contenant 10% de sérum fœtal bovin (FBS ), de pénicilline-streptomycine (pénicilline: 100 U / ml, streptomycine: 100 pg / ml) (Invitrogen, Cat # 15140122), et 600 pg / ml d&#39;hygromycine B ...

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Systèmes de génétique inverse pour les RVFV ont été développés par plusieurs groupes en utilisant le promoteur T7 2,4,5 ou une souris 3 ou 4 humains Pol-i promoteur. Dans ce manuscrit, nous décrivons un protocole visant à générer des recombinants RVFV MP-12 souches en utilisant BHK/T7-9 cellules 15 qui expriment de façon stable la T7 RNA polymérase. L&#39;efficacité de la récupération virale varie en fonction de l&#39;état de BHK/T7-9 cellules, le montant des ...

<table border="1"><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Minimum Essential Medium (MEM)-alpha </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Modifié par Dulbecco milieu essentiel minimum </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> Pénicilline-streptomycine </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> Hygromycine B </td><td> Cellgro </td><td> 30 à 240-CR </td><td></td></tr><tr><td> Bouillon tryptose phosphate </td><td> MP Biomédical </td><td> 1682149 </td><td></td></tr><tr><td> Gélose 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> Aérosol couvercle étanche </td><td> Eppendorf </td><td> C-2223-25 </td><td></td></tr><tr><td> 0,33% de solution de rouge neutre </td><td> Sigma-Aldrich </td><td> N2889-100ML </td><td></td></tr><tr><td> C57/WT cellules MEF </td><td> InvivoGen </td><td> MEF-c57wt </td><td></td></tr><tr><td> Blasticidine S </td><td> InvivoGen </td><td> Ant-bl-1 </td><td></td></tr><tr><td> Zéocine </td><td> InvivoGen </td><td> ANT-zn-1 </td><td></td></tr><tr><td> Quanti-Bleu </td><td> InvivoGen </td><td> rep-QB1 </td><td></td></tr><tr><td> BHK/T7-9 cellules 15 </td><td> Université de Gifu, au Japon </td><td></td><td></td></tr><tr><td> Cellules Vero E6 </td><td> ATCC </td><td> LCR-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 de la fièvre vallée du Rift (FVR), qui provoque une fièvre hémorragique, des troubles neurologiques ou de cécité chez les humains, et un taux élevé d&#39;avortement et de malformations fœtales chez les ruminants 1, a été classé comme un HHS / USDA chevauchent agent de sélection et un groupe de risque 3 pathogène. Il appartient au genre Phlebovirus de la famille des Bunyaviridae et est l&#39;un des membres les plus virulents de cette famille. Plusieurs systèmes de génétique inverse pour la souche RVFV MP-12 2,3 vaccins ainsi que des souches de type sauvage RVFV 4-6, y compris les ZH548 ZH501 et ont été développés depuis 2006. Le MP-12 souche (qui est un groupe de risque 2 et un agent pathogène non-sélection) est fortement atténué par plusieurs mutations dans son M-et L-segments, mais porte toujours virulentes S-segment d&#39;ARN 3, qui code une virulence fonctionnelle facteur, NSS. Le rMP12-C13type (C13type) transportant 69% dans le châssis-suppression de NSS ORF manque toutes les fonctions connues NSs, tandis qu&#39;il réplique que efficient tout comme MP-12 dans des cellules dépourvues VeroE6 type I IFN. NSs induit un arrêt de la transcription de l&#39;hôte dont l&#39;interféron (IFN)-bêta ARNm 7,8 et favorise la dégradation de la double-brin protéine kinase dépendante de l&#39;ARN (PKR) au niveau post-traductionnelles. 9,10 IFN-bêta est transcriptionnellement régulés par interferon regulatory factor 3 (IRF-3), NF-kB et la protéine activatrice-1 (AP-1), et la liaison de l&#39;IFN-bêta à IFN-alpha/beta récepteur (IFNAR) stimule la transcription de l&#39;IFN-alpha gènes ou d&#39;autres gènes d&#39;interféron stimulée (ISGS) 11, ce qui induit des activités d&#39;accueil antiviraux, alors que la suppression de la transcription d&#39;hôte incluant l&#39;IFN-bêta du gène par le NSS empêche le gène de ceux upregulations ISGS en réponse à la réplication virale, bien que l&#39;IRF-3, NF-kB et l&#39;activateur protein-1 (AP-1) peut être activé par RVFV7. . Ainsi, NSS est une excellente cible pour atténuer encore MP-12, et d&#39;améliorer la réponse immunitaire innée de l&#39;hôte en abolissant la fonction de suppression de l&#39;IFN-bêta. Voici, Nous décrivons un protocole pour générer un SSN-12 recombinante MP encodage muté, et fournir un exemple d&#39;une méthode de dépistage pour identifier les mutants NSs manque la fonction pour supprimer synthèse de l&#39;ARNm de l&#39;IFN-bêta. En plus de son rôle essentiel dans l&#39;immunité innée, l&#39;IFN de type I est important pour la maturation des cellules dendritiques et l&#39;induction d&#39;une réponse immunitaire adaptative 12-14. Ainsi, les mutants NSs induisant l&#39;IFN de type I sont plus atténuées, mais en même temps, sont plus efficaces pour stimuler des réponses immunitaires que l&#39;hôte de type sauvage MP-12, qui fait des candidats idéaux pour des approches de vaccination.

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

Le système de génétique inverse pour le virus de la fièvre vallée du Rift MP-12 souche vaccinale est un outil utile pour créer des supplémentaires MP-12 mutants avec une atténuation accrue et l&#39;immunogénicité. Nous décrivons le protocole de générer et caractériser NSs souches mutantes.

En utilisant la génétique inverse pour manipuler les gènes NSS du virus de la fièvre vallée du Rift MP-12 souche pour améliorer la sécurité vaccinale et l&#39;efficacité

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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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

L’Assemblée et la Purification du Prototype spumavirus Intasomes

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

Évaluation des réponses de l’hôte-pathogène et l’efficacité du vaccin chez la souris

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