रिवर्स आनुवंशिकी प्रणाली दरार घाटी बुखार वायरस सांसद 12 टीका तनाव के लिए बढ़ क्षीणन और प्रतिरक्षाजनकता साथ अतिरिक्त सांसद 12 म्यूटेंट बनाने के लिए एक उपयोगी उपकरण है. हम प्रोटोकॉल का वर्णन करने के लिए पैदा करते हैं और एनएसएस उत्परिवर्ती उपभेदों विशेषताएँ.
Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.
RVFV के लिए आनुवंशिकी सिस्टम उल्टा T7 प्रमोटर 2,4,5 या 3 माउस या मानव 4 प्रमोटर नीति मैं का उपयोग करके कई समूहों द्वारा विकसित किया गया है . इस पांडुलिपि में, हम BHK/T7-9 15 कि stably व्यक्त T7 शाही सेना पोलीम?…
The authors have nothing to disclose.
इस काम अनुदान 5 नंबर U54-07 उत्कृष्टता के पश्चिमी क्षेत्रीय केंद्र (WRCE) के माध्यम से AI057156, नेशनल इंस्टीट्यूट ऑफ एलर्जी और संक्रामक रोगों से 1 AI08764301 – A1 R01, और विश्वविद्यालय में एक टीके के विकास के लिए Sealy केंद्र से आंतरिक धन के द्वारा वित्त पोषित किया गया था टेक्सास चिकित्सा शाखा है.
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 |