Brug af omvendt genetik til at manipulere NSS Gene af Rift Valley Fever virus MP-12 Strain to Improve Vaccine sikkerhed og effekt
Summary
Det omvendte genetik system for Rift Valley fever virus MP-12 vaccinestamme er et nyttigt værktøj til at skabe ekstra MP-12 mutanter med øget dæmpning og immunogenicitet. Vi beskriver protokollen til at generere og karakterisere NSS mutant stammer.
Abstract
Rift Valley fever virus (RVFV), som forårsager hæmoragisk feber, neurologiske lidelser eller blindhed hos mennesker, og en høj abort og fosterskader hos drøvtyggere 1 er blevet klassificeret som et HHS / USDA overlap vælge agent og en risiko gruppe 3 patogen. Den tilhører slægten Phlebovirus i familien Bunyaviridae og er en af de mest virulente medlemmerne af denne familie. Flere omvendt genetik systemer til RVFV MP-12 vaccinestamme 2,3 samt vildtype RVFV stammer 4-6, herunder ZH548 og ZH501, er blevet udviklet siden 2006. MP-12 stamme (som er en risiko gruppe 2 patogen og en ikke-vælg agent) er stærkt svækket af flere mutationer i sin M-og L-segmenter, men stadig bærer virulent S-segmentet RNA 3, som koder for en funktionel virulens faktor, NSS. Den rMP12-C13type (C13type) transporterer 69% i-frame sletning af NSS ORF mangler alle de kendte NSS funktioner, mens det replikater som efficient som gør MP-12 i VeroE6 celler mangler type I-IFN. NSS inducerer en afspærring af vært transskription herunder interferon (IFN)-beta mRNA 7,8 og fremmer nedbrydning af dobbelt-strenget RNA-afhængig protein kinase (PKR) på post-translationel niveau. 9,10 IFN-beta er transcriptionally opreguleres med interferon regulerende faktor 3 (IRF-3), NF-kB og aktivator protein-1 (AP-1), og bindingen af IFN-beta til IFN-alpha/beta receptor (IFNAR) stimulerer transskription af IFN-alpha gener eller andre interferon stimuleret gener (ISGs) 11, som fremkalder vært antivirale aktiviteter, mens værten transskription undertrykkelse herunder IFN-beta-genet fra NSS forhindrer genet upregulations af disse ISGs som reaktion på virusreplikation selvom IRF-3, NF-kB og aktivator protein-1 (AP-1) kan aktiveres ved RVFV7. . Således NSS er et fremragende mål til yderligere at dæmpe MP-12, og for at forbedre vært medfødte immunrespons ved at afskaffe den IFN-beta undertrykkelse funktion. Her, Vi beskriver en protokol for at generere en rekombinant MP-12 kodning muterede NSS, og giver et eksempel på en screeningsmetode til at identificere NSS mutanter mangler den funktion at undertrykke IFN-beta mRNA syntese. Ud over sin afgørende rolle i medfødte immunitet, er type-I IFN betydning for modningen af dendritiske celler og induktion af et adaptivt immunrespons 12-14. Således NSS mutanter overtalelse type I-IFN er yderligere svækket, men samtidig er mere effektive til at stimulere vært immunrespons end vildtype-MP-12, hvilket gør dem ideelle kandidater til vaccination tilgange.
Protocol
Discussion
Omvendt genetik systemer til RVFV er udviklet af flere grupper ved at udnytte T7 promotoren 2,4,5 eller mus 3 eller menneskelige 4 pol-I promotor. I dette manuskript, beskriver vi en protokol til at generere rekombinant RVFV MP-12 stammer ved hjælp BHK/T7-9 celler 15, der stabilt udtrykker T7 RNA polymerase. Effektiviteten af virale inddrivelsen forskellig, afhængig af tilstand BHK/T7-9 celler, mængden af plasmider, antallet af transfekterede celler og så vider…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev finansieret af Grant Nummer 5 U54 AI057156-07 via Western Regional Center of Excellence (WRCE), 1 R01 AI08764301-A1 fra National Institute of Allergy og infektionssygdomme, og en intern finansiering fra Sealy Center for Vaccine udvikling på University of Texas Medical Branch.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Minimum Essential Medium (MEM)-alpha | Invitrogen | 32561037 | |
| Dulbecco’s modified minimum essential medium | Invitrogen | 11965092 | |
| Modified Eagle Medium (MEM 2x) | Invitrogen | 11935046 | |
| Penicillin-Streptomycin | Invitrogen | 15140122 | |
| Hygromycin B | Cellgro | 30-240-CR | |
| Tryptose phosphate broth | MP biomedicals | 1682149 | |
| Noble agar | VWR | 101170-362 | |
| TransIT-LT1 | Mirus | MIR2300 | |
| Opti-MEM | Invitrogen | 31985070 | |
| Aerosol tight lid | Eppendorf | C-2223-25 | |
| 0.33% neutral red solution | Sigma Aldrich | N2889-100ML | |
| C57/WT MEF cells | InvivoGen | mef-c57wt | |
| Blasticidin S | InvivoGen | Ant-bl-1 | |
| Zeocin | InvivoGen | ant-zn-1 | |
| QUANTI-Blue | InvivoGen | rep-qb1 | |
| BHK/T7-9 cells15 | Gifu university, Japan | ||
| Vero E6 cells | ATCC | CRL-1586 |
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