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

Dette arbeidet ble finansiert av Grant Nummer 5 U54 AI057156-07 gjennom Western Regional Center of Excellence (WRCE), 1 R01 AI08764301-A1 fra National Institute of Allergy og smittsomme sykdommer, og en intern finansiering fra Sealy Center for Vaccine Development ved Universitetet i Texas Medical Branch.

1. Gjenoppretting av rekombinant MP-12 encoding NSS mutasjon (s) fra plasmid DNAS 2 <ol><li> Spre hamsterunge nyre (BHK) / T7-9 celler 15, som stabilt uttrykke T7 RNA polymerase, inn 6-cm retter i Minimum Essential Medium (MEM)-alpha (Invitrogen, Cat # 32561037) inneholder 10% fetal bovint serum (FBS ), Penicillin-Streptomycin (Penicillin: 100 U / ml, Streptomycin: 100 mikrogram / ml) (Invitrogen, Cat # 15140122), og 600 mikrogram / ml av hygromycin B (Cellgro, Cat # 30 x 240 CR). * Effekt...

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Revers genetikk systemer for RVFV har blitt utviklet av flere grupper ved å utnytte T7 promoter 2,4,5 eller mus 3 eller human fire pol-I promoter. I dette manuskriptet beskriver vi en protokoll for å generere rekombinant RVFV MP-12 stammer ved hjelp BHK/T7-9 celler 15 som stabilt uttrykke T7 RNA polymerase. Effektiviteten av viral utvinning varierte avhengig av tilstanden til BHK/T7-9 celler, mengden av plasmider, antall transfektert celler og så videre. Vi har alltid forst...

<table border="1"><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfritt) </td></tr><tr><td> Minimum Essential Medium (MEM)-alfa </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Dulbecco modifiserte minimum viktig medium </td><td> Invitrogen </td><td> 11965092 </td><td></td></tr><tr><td> Modifisert Eagle Medium (MEM 2x) </td><td> Invitrogen </td><td> 11935046 </td><td></td></tr><tr><td> Penicillin-Streptomycin </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 kjøttkraft </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 tett lokk </td><td> Eppendorf </td><td> C-2223-25 </td><td></td></tr><tr><td> 0,33% nøytral rød-løsning </td><td> Sigma Aldrich </td><td> N2889-100ML </td><td></td></tr><tr><td> C57/WT MEF celler </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> Mengdene Blå </td><td> InvivoGen </td><td> rep-QB1 </td><td></td></tr><tr><td> BHK/T7-9 celler 15 </td><td> Gifu University, Japan </td><td></td><td></td></tr><tr><td> Vero E6 celler </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

Rift Valley fever virus (RVFV), som forårsaker hemoragisk feber, nevrologiske lidelser eller blindhet hos mennesker, og en høy abort og fosterets misdannelser hos drøvtyggere 1, har blitt klassifisert som et HHS / USDA overlapping velge agent og en risikogruppe 3 patogen. Den tilhører slekten Phlebovirus i familien Bunyaviridae og er en av de mest virulente medlemmene av denne familien. Flere revers genetikk systemer for RVFV MP-12 vaksinestammen 2,3 samt villtype RVFV stammer 4-6, herunder ZH548 og ZH501, har blitt utviklet siden 2006. MP-12 belastning (som er en risiko gruppe 2 patogen og en ikke-velg agent) er svært svekket av flere mutasjoner i M-og L-segmenter, men likevel bærer virulente S-segment 3 RNA, som koder for et funksjonelt virulens faktor, NSS. Den rMP12-C13type (C13type) bærer 69% i-frame sletting av NSS ORF mangler alle de kjente NSS funksjoner, mens det replikeres som efficient som gjør MP-12 i VeroE6 celler mangler type-I IFN. NSS induserer en avstenging av verten transkripsjon herunder interferon (IFN)-beta mRNA 7,8 og fremmer nedbrytning av dobbel-strandet RNA-avhengig protein kinase (PKR) på posttranslasjonelle nivå. 9,10 IFN-beta er transcriptionally oppregulert med interferon regulerende faktor 3 (IRF-3), NF-kB og aktivator protein-1 (AP-1), og bindingen av IFN-beta til IFN-alpha/beta reseptor (IFNAR) stimulerer transkripsjon av IFN-alfa gener eller andre interferon stimulert gener (ISGs) 11, som induserer vert antiviral aktiviteter, mens host transkripsjon undertrykkelse inkludert IFN-beta genet ved NSS hindrer genet upregulations av de ISGs svar på virusreplikasjon selv om IRF-3, NF-kB og aktivator protein-1 (AP-1) kan aktiveres ved RVFV7. . Dermed er NSS et utmerket mål for ytterligere å dempe MP-12, og for å styrke host medfødte immunresponser ved å avskaffe IFN-beta undertrykkelse funksjon. HerBeskriver vi en protokoll for å generere et rekombinant MP-12 koding mutert NSS, og gi et eksempel på en screening metode for å identifisere NSS mutanter mangler funksjon for å undertrykke IFN-beta mRNA syntese. I tillegg til sin avgjørende rolle i medfødt immunitet, er type-I IFN viktig for modningen av dendrittiske celler og induksjon av en adaptiv immunrespons 12-14. Dermed NSS mutanter inducing type-I IFN er ytterligere svekket, men samtidig er mer effektiv på å stimulere vert immunresponser enn vill-type MP-12, noe som gjør dem ideelle kandidater for vaksinasjon tilnærminger.

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

Den revers genetikk system for Rift Valley fever virus MP-12 vaksinestammen er et nyttig verktøy for å skape ekstra MP-12 mutanter med økt demping og immunogenisitet. Vi beskriver protokollen til å generere og karakterisere NSS mutant stammer.

Ved hjelp av revers genetikk å manipulere NSS Gene av Rift Valley Fever Virus MP-12 Strain å forbedre Vaccine sikkerhet og effekt

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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