Diese Arbeit wurde durch Grant Number 5 U54 AI057156-07 durch Western Regional Center of Excellence (WRCE), 1 R01 AI08764301-A1 von National Institute of Allergy and Infectious Diseases, und eine interne Finanzierung von Sealy Center for Vaccine Development an der Universität finanziert Texas Medical Branch.

<ol>
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Wir haben nichts zu offenbaren.

Die reverse Genetik für RVFV wurden von mehreren Gruppen wurden durch den Einsatz T7-Promotor 2,4,5 oder Maus 3 oder menschliche 4 pol-I-Promotor entwickelt. In diesem Manuskript, beschreiben wir ein Protokoll zur rekombinanten RVFV MP-12-Stämme mit BHK/T7-9 Zellen 15, die stabil exprimieren, T7-RNA-Polymerase zu erzeugen. Die Effizienz der viralen Erholung variiert je nach Zustand des BHK/T7-9 Zellen, die Menge von Plasmiden, die Anzahl der transfizierten Zellen und so weit...

<table border="1"><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Minimum Essential Medium (MEM)-alpha </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Dulbecco modifiziertem Minimum Essential Medium </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> Penicillin-Streptomycin </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> Hygromycin B </td><td> CellGro </td><td> 30 bis 240-CR </td><td></td></tr><tr><td> Tryptosephosphat Brühe </td><td> MP Biomedicals </td><td> 1682149 </td><td></td></tr><tr><td> Edle 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 dichten Deckel </td><td> Eppendorf </td><td> C-2223 bis 25 </td><td></td></tr><tr><td> 0,33% Neutralrot-Lösung </td><td> Sigma Aldrich </td><td> N2889-100ML </td><td></td></tr><tr><td> C57/WT MEF-Zellen </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 Zellen 15 </td><td> Gifu University, Japan </td><td></td><td></td></tr><tr><td> Vero E6-Zellen </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-Fieber-Virus (RVFV), die hämorrhagisches Fieber, neurologischen Erkrankungen oder Erblindung bei Menschen, und eine hohe Rate Abtreibung und fetale Missbildungen bei Wiederkäuern 1 bewirkt hat als HHS / USDA eingestuft überschneiden wählen Agent und eine Risikogruppe 3 Erreger. Es gehört zur Gattung Phlebovirus in der Familie Bunyaviridae und ist einer der bösartigsten Mitglieder dieser Familie. Mehrere reversen Genetik-Systeme für den RVFV MP-12 Impfstamm 2,3 sowie Wildtyp-RVFV Stämme 4-6, einschließlich ZH548 und ZH501, seit 2006 entwickelt worden. Der MP-12-Stamm (das ist ein Risiko, Gruppe 2 Erreger und einem Nicht-wählen-Agent) ist stark gedämpft durch mehrere Mutationen im M-und L-Segmente, aber trägt noch virulent S-Segment RNA 3, die eine funktionelle Virulenz kodiert Faktor, NSS. Die rMP12-C13type (C13type) tragen 69% in-frame-Deletion von NSS ORF fehlt allen bekannten NSs Funktionen, während es als effic repliziertient ebenso wie MP-12 in VeroE6 Zellen ohne Typ-I-IFN. NSs induziert eine Abschaltung von Host-Transkription einschließlich Interferon (IFN)-beta mRNA 7,8 und fördert Abbau von doppelsträngigen RNA-abhängige Proteinkinase (PKR) in der post-translationalen Ebene. 9,10 IFN-beta ist transkriptionell hochreguliert durch Interferon regulatory factor 3 (IRF-3), NF-kB und Aktivator-Protein-1 (AP-1), und die Bindung von IFN-beta zu IFN-alpha/beta Rezeptor (IFNAR) stimuliert die Transkription von IFN-alpha Gene oder anderen Interferon-stimulierten Genen (ISGs) 11, welcher Host antivirale Aktivitäten induziert, während Host Transkription Unterdrückung einschließlich IFN-beta-Gen von NSS verhindert das Gen upregulations dieser ISGs in Reaktion auf die virale Replikation, obwohl IRF-3, NF-kB und Aktivator Protein-1 (AP-1) kann durch RVFV7 aktiviert werden. . So ist NSs ein hervorragendes Ziel weiter dämpfen MP-12 und zum Host angeborenen Immunantwort durch die Abschaffung der IFN-beta Unterdrückung Funktion zu verbessern. HierWir beschreiben ein Protokoll zur Erzeugung eines rekombinanten MP-12-Codierung mutierte NSS und liefern ein Beispiel für ein Screening-Verfahren auf NSS-Mutanten fehlt die Funktion, IFN-beta-mRNA-Synthese zu unterdrücken identifizieren. Zusätzlich zu ihrer wesentlichen Rolle in der angeborenen Immunität, ist der Typ-I IFN für die Reifung von dendritischen Zellen und die Induktion einer adaptiven Immunantwort 12-14 wichtig. So induzieren NSs Mutanten-Typ-I IFN sind weiter abgeschwächt, aber gleichzeitig sind effizienter zu stimulieren Host Immunreaktionen als Wildtyp-MP-12, die sie zu idealen Kandidaten für die Impfung Ansätze macht.

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

Die reverse Genetik für die Rift Valley-Fieber-Virus MP-12 Impfstamm ist ein nützliches Werkzeug für die Schaffung zusätzlicher MP-12-Mutanten mit erhöhter Dämpfung und Immunogenität. Wir beschreiben das Protokoll zu erzeugen und zu charakterisieren NSs Mutanten.

Mit Reverse-Genetics der NSS Gene des Rift Valley Fever Virus MP-12-Stamm zu Vaccine Safety und Wirksamkeit zu verbessern Manipulieren

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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Immunology and Infection

17.2K Aufrufe.  University of Texas Medical Branch. Die reverse Genetik für die Rift Valley-Fieber-Virus MP-12 Impfstamm ist ein nützliches Werkzeug für die Schaffung zusätzlicher MP-12-Mutanten mit erhöhter Dämpfung und Immunogenität. Wir beschreiben das Protokoll zu erzeugen und zu charakterisieren NSs Mutanten.