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

Este trabalho foi financiado pelo Grant Number 5 U54 AI057156-07 através do Centro Regional de Excelência Ocidental (WRCE), um R01 AI08764301-A1 do Instituto Nacional de Alergia e Doenças Infecciosas, e um financiamento interno do Sealy Center for Vaccine Development da Universidade de Texas Medical Branch.

1. Recuperação de recombinantes mutação NSS MP-12 codificação (s) de DNAs plasmídeo 2 <ol><li> Spread rim de hamster bebé (BHK) / T7-9 células 15, que expressa estavelmente T7 RNA polimerase, em 6 cm de pratos em Meio Essencial Mínimo (MEM)-alfa (Invitrogen, Cat # 32561037) contendo 10% de soro fetal bovino (FBS ), penicilina-estreptomicina (Penicilina: 100 U / ml Estreptomicina: 100 mcg / ml) (Invitrogen, Cat # 15140122), e 600 mg / ml de higromicina B (Cellgro, Cat # 30-240-CR). <br /...

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Genética reversa de sistemas para RVFV foram desenvolvidos por vários grupos, utilizando T7 promoter 2,4,5 ou mouse de 3 ou 4 humanos promotor pol-I. Neste manuscrito, nós descrevemos um protocolo para gerar recombinantes RVFV MP-12 linhagens de células usando BHK/T7-9 15 que expressa estavelmente T7 RNA polimerase. A eficiência de recuperação viral variava de acordo com a condição de BHK/T7-9 células, a quantidade de plasmídeos, o número de células transfectadas ...

<table border="1"><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comments (opcional) </td></tr><tr><td> Meio Essencial Mínimo (MEM)-alfa </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Dulbecco modificado de médio mínimo essencial </td><td> Invitrogen </td><td> 11965092 </td><td></td></tr><tr><td> Modificado Águia Medium (MEM 2x) </td><td> Invitrogen </td><td> 11935046 </td><td></td></tr><tr><td> Penicilina-estreptomicina </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> Higromicina B </td><td> Cellgro </td><td> 30-240-CR </td><td></td></tr><tr><td> Triptose fosfato de caldo </td><td> Biomedicals MP </td><td> 1682149 </td><td></td></tr><tr><td> Ágar Noble </td><td> VWR </td><td> 101170-362 </td><td></td></tr><tr><td> Trânsito 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> Tampa apertada Aerosol </td><td> Eppendorf </td><td> C-2223-25 </td><td></td></tr><tr><td> 0,33% solução de vermelho neutro </td><td> Sigma Aldrich </td><td> N2889-100ML </td><td></td></tr><tr><td> Células C57/WT 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-Blue </td><td> InvivoGen </td><td> rep-QB1 </td><td></td></tr><tr><td> BHK/T7-9 células 15 </td><td> Gifu University, Japão </td><td></td><td></td></tr><tr><td> Células Vero E6 </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

Vírus da febre Rift Valley (RVFV), que causa febre hemorrágica, distúrbios neurológicos ou cegueira em humanos, e uma alta taxa de aborto e malformação fetal em ruminantes 1, foi classificado como um HHS / USDA sobreposição agente selecionado e um grupo de risco 3 patógeno. Pertence à Phlebovírus gênero no Bunyaviridae família e é um dos membros mais virulenta dessa família. Vários sistemas de genética reversa para a cepa vacinal MP-12 RVFV 2,3, bem como cepas do tipo selvagem RVFV 4-6, incluindo ZH548 e ZH501, têm sido desenvolvidos desde 2006. O MP-12 estirpe (que é um grupo de risco 2 patógeno e um agente não-select) é altamente atenuada por várias mutações em seu M e L-segmentos, mas ainda carrega virulenta segmento S-RNA 3, que codifica uma virulência funcional NSS fator. O rMP12-C13type (C13type) transportando 69% em-frame de eliminação do NSS ORF não tem todas as funções NSS conhecida, enquanto ele replica como effictário como faz MP-12 em células VeroE6 faltando tipo I IFN. NSS induz um shut-off de transcrição de hospedeiros, incluindo interferon (IFN)-beta mRNA 7,8 e promove a degradação de double-stranded RNA-proteína quinase dependente (PKR) em nível de pós-translacionais. 9,10 IFN-beta é transcricionalmente upregulated por interferon fator de regulação 3 (IRF-3), NF-kB e proteína ativadora-1 (AP-1), ea ligação do IFN-beta para IFN-alpha/beta receptor (IFNAR) estimula a transcrição de IFN-alpha genes ou de outros genes interferon estimulada (ISGs) 11, que induz actividades de acolhimento antiviral, ao passo que a supressão anfitrião de transcrição incluindo IFN-beta gene pela NSS impede que o gene upregulations daqueles ISGs em resposta a replicação viral, embora IRF-3, NF-kB e ativador proteína-1 (AP-1) pode ser ativado por RVFV7. . Assim, o NSS é um excelente alvo para continuar a atenuar MP-12, e para melhorar a resposta imune inata anfitrião, abolindo a função de supressão de IFN-beta. Aqui, Nós descrevemos um protocolo para a geração de um recombinante MP-12 NSS codificação mutado, e fornecer um exemplo de um método de triagem para identificar mutantes NSS falta a função de suprimir a síntese de IFN-beta mRNA. Além de seu papel essencial na imunidade inata, tipo I IFN é importante para a maturação das células dendríticas e indução de uma resposta imune adaptativa 12-14. Assim, os mutantes NSS induzindo tipo I IFN são mais atenuadas, mas ao mesmo tempo, são mais eficientes na resposta do hospedeiro estimulante imunológico do tipo selvagem MP-12, o que os torna candidatos ideais para abordagens de vacinação.

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

O sistema de genética reversa para o vírus da febre Rift Vale cepa vacinal MP-12 é uma ferramenta útil para a criação de adicionais MP-12 mutantes com atenuação aumentada e imunogenicidade. Nós descrevemos o protocolo para gerar e caracterizar NSS cepas mutantes.

Usando genética reversa para manipular o NSS Gene do vírus da febre Rift Valley MP-12 Strain para melhorar a segurança e eficácia da vacina

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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