Name: analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps
Uploaded: 2013-02-14T12:00:00
Description: Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

1. Virus Inokulation og VLP&#39;er Oprensning fra Nicotiana benthamiana Planter <ol><li> Fremstille udjævnet T7-RNA-transkripter fra kartoffel-virus X (PVX)-baseret vektor plasmider, der bærer MRFV vildtype (wt) og Cys-muterede kappeprotein (CP) generne 12, ved hjælp af Ambion s T7-mMessage mMachine Kit. </li><li> For hver T7 transcript reaktion, podes to fuldstændigt udfoldede blade af N. benthamiana med 10 gl reaktioner og inkuberes planterne i 10 dage i drivhus ved 60% fugt...

<ol>
	<li>Barnhill, H., Reuther, R., Ferguson, P. L., Dreher, T. W., Wang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnip+yellow+mosaic+virus+as+a+chemoaddressable+bionanoparticle.">Turnip yellow mosaic virus as a chemoaddressable bionanoparticle.</a> Bioconj. Chem. 18, 852-859 (2007).</li><li>Chapman, S., Kavanagh, T., Baulcombe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+virus+X+as+a+vector+for+gene+expression+in+plants.">Potato virus X as a vector for gene expression in plants.</a> Plant J. 2, 549-557 (1992).</li><li>Chen, C., Kwak, E. S., Stein, B., Kao, C. C., Dragnea, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+of+gold+particles+in+viral+capsids.">Packaging of gold particles in viral capsids.</a> J. Nanosci. Nanotechnol. 5, 2029-2033 (2005).</li><li>Fowler, C. E., Shenton, W., Stubbs, G., Mann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+liquid+crystals+as+templates+for+the+interior+design+of+silica+mesophases+and+nanoparticles.">Tobacco mosaic virus liquid crystals as templates for the interior design of silica mesophases and nanoparticles.</a> Advanced Materials. 13, 1266-1269 (2001).</li><li>Gazit, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biomolecular+templates+for+the+fabrication+of+metal+nanowires.">Use of biomolecular templates for the fabrication of metal nanowires.</a> FEBS. J. 274, 317-322 (2007).</li><li>Gillitzer, E., Wilts, D., Young, M., Douglas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+of+a+viral+cage+for+multivalent+presentation.">Chemical modification of a viral cage for multivalent presentation.</a> Chem. Commun. , 2390-2391 (2002).</li><li>Hammond, R. W., Hammond, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+rayado+fino+virus+capsid+proteins+assemble+into+virus-like+particles+in+Escherichia+coli.">Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli.</a> Virus Res. 147, 208-215 (2010).</li><li>Hermamson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate techniques. , (1991).</li><li>Kaiser, C. R., Flenniken, M. L., Gillitzer, E., Harmsen, A. L., Harmsen, A. G., Jutila, M. A., Douglas, T., Young, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodistribution+studies+of+protein+cage+nanoparticles+demonstrate+broad+tissue+distribution+and+rapid+clearance+in+vivo.">Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo.</a> Int. J. Nanomed. 2, 715-733 (2007).</li><li>Knez, M., Bittner, A. M., Boes, F., Wege, C., Jeske, H., Maisse, E., Kern, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotemplate+synthesis+of+3-nm+nickel+and+cobalt+nanowires.">Biotemplate synthesis of 3-nm nickel and cobalt nanowires.</a> Nano Lett. 3, 1079-1082 (2003).</li><li>Lee, S. Y., Culver, J. N., Harris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+CuCl2+concentration+on+the+aggregation+and+mineralization+of+Tobacco+mosaic+virus+biotemplate.">Effect of CuCl2 concentration on the aggregation and mineralization of Tobacco mosaic virus biotemplate.</a> J. Colloid. Interface. Sci. 297, 554-560 (2006).</li><li>Lvov, Y., Haas, H., Decher, G., Mohwald, H., Mikhailov, A., Mtchedlishvily, B., Morgunova, E., Vainshtein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successive+deposition+of+alternate+layers+of+polyelectrolytes+and+a+charged+virus.">Successive deposition of alternate layers of polyelectrolytes and a charged virus.</a> Langmuir. 10, 4232-4236 (1994).</li><li>Natilla, A., Hammond, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+rayado+fino+virus+virus-like+particles+expressed+in+tobacco+plants:+a+new+platform+for+cysteine+selective+bioconjugation+peptide+display.">Maize rayado fino virus virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display.</a> J. Virol. Methods. 178, 209-215 (2011).</li><li>Rae, C. S., Khor, I. W., Wang, Q., Destito, G., Gonzalez, M. J., Singh, P., Thomas, D. M., Estrada, M. N., Powell, E., Finn, M. G., Manchester, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+trafficking+of+plant+virus+nanoparticles+in+mice+via+the+oral+route.">Systemic trafficking of plant virus nanoparticles in mice via the oral route.</a> Virology. 343, 2224-2235 (2005).</li><li>Raja, K. S., Wang, Q., Gonzalez, M. J., Manchester, M., Johnson, J. E., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+virus-polymer+materials.+Synthesis+and+properties+of+PEG-decorated+Cowpea+mosaic+virus.">Hybrid virus-polymer materials. Synthesis and properties of PEG-decorated Cowpea mosaic virus.</a> Biomacromolecules. 4, 472-476 (2003).</li><li>Royston, E., Lee, S. Y., Culver, J. N., Harris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silica-coated+Tobacco+mosaic+virus.">Characterization of silica-coated Tobacco mosaic virus.</a> J. Colloid Interface Sci. 298, 706-712 (2006).</li><li>Schlick, T. L., Ding, Z., Kovacs, E. W., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-surface+modification+in+the+Tobacco+mosaic+virus.">Dual-surface modification in the Tobacco mosaic virus.</a> J. Am. Chem. Soc. 127, 3718-3723 (2005).</li><li>Young, M., Willits, D., Uchida, M., Douglas, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+as+biotemplates+for+materials+and+their+use+in+nanotechnology.">Plant viruses as biotemplates for materials and their use in nanotechnology.</a> Annu. Rev. Phytopathol. 46, 361-384 (2008).</li></ol>

Fremgangsmåden præsenteres her muliggør en meget følsom og hurtig analyse af reaktive cysteiner er til stede på overfladen af ​​plante-produceret VLP&#39;er samt andre protein-komplekser. Maleimider er thiol-specifikke reagenser, som reagerer med frie sulfhydrylholdige molekyler til dannelse af stabile thioetherbindinger. Denne metode bygger på specificitet maleimider til at reagere med sulfhydrylgrupper ikke er involveret i samspillet med andre aminosyrer. Bevarer den native struktur af VLP&#39;erne er meget ...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Thinwall, Ultra-Clear Tubes </td><td> Beckman </td><td> 344.059 </td><td></td></tr><tr><td> mMESSAGE mMACHINE T7 Kit </td><td> Life Tecnologies </td><td> AM1344M </td><td></td></tr><tr><td> Fluorescein-5-maleimid </td><td> Thermo Scientific Life Technologies </td><td> 46.130 F150 </td><td> 46.130 er ude af drift erstatning med F150 </td></tr><tr><td> Pierce Biotin Kvantificering Kit </td><td> Thermo Scientific </td><td> 28.005 </td><td></td></tr><tr><td> EZ-Link maleimid-PEG2-biotin, No-afvejes Format </td><td> Thermo Scientific </td><td> 21.901 </td><td></td></tr><tr><td> SM (PEG) n Tværbindere </td><td> Thermo Scientific </td><td> 22.107 </td><td></td></tr><tr><td> 10-20% Tris-Glycin gel </td><td> Invitrogen </td><td> EC61352 </td><td></td></tr><tr><td> Laemmli puffer </td><td> Bio-Rad </td><td> 1610737 </td><td></td></tr><tr><td> Tris Glycine SDS Running Buffer </td><td> Invitrogen </td><td> LC2675 </td><td></td></tr><tr><td> Tris Glycine overførselspuffer </td><td> Invitrogen </td><td> LC3675 </td><td></td></tr><tr><td> Nitrocellulosemembran Filter Paper Sandwich </td><td> Invitrogen </td><td> LC2001 </td><td></td></tr><tr><td> Phosphatase-mærket affinitetsoprenset antistof mod kanin-IgG </td><td> Kirkegaard and Perry Laboratories </td><td> 0751516 </td><td></td></tr><tr><td> NBT / BCIP phosphatasesubstrat </td><td> Kirkegaard and Perry Laboratories </td><td> 508.107 </td><td></td></tr></tbody></table>

analysis of the solvent accessibility of cysteine residues on maize rayado fino virus virus-like particles produced in nicotiana benthamiana plants and cross-linking of peptides to vlps

Efterligning og udnytte virus egenskaber og fysisk-kemiske og fysiske egenskaber besidder løfte om at levere løsninger til nogle af verdens mest presserende udfordringer. Alene rækkevidde og virustyper kombineret med deres spændende egenskaber potentielt give uendelige muligheder for ansøgninger i virus-baserede teknologier. Vira har evnen til at selv-samle til partikler med diskrete form og størrelse, specificitet symmetri, alsidighed og stabile egenskaber under et bredt område af temperatur-og pH-betingelser. Ikke overraskende med sådan en bemærkelsesværdig vifte af egenskaber, er virus foreslået til anvendelse i biomaterialer 9, vacciner 14, 15, elektroniske materialer, kemiske værktøjer og molekylær elektronisk beholdere 4, 5, 10, 11, 16, 18, ​​12. For at udnytte virus inden for nanoteknologi, skal de ændres fra deres naturlige former for at give nye funktioner. Denne udfordrende process kan udføres gennem flere mekanismer, herunder genetisk modifikation af det virale genom og kemisk fastgørelse af fremmede eller ønskede molekyler til viruspartiklen reaktive grupper 8. Evnen til at ændre en virus primært afhænger af de fysisk-kemiske og fysiske egenskaber af viruset. Desuden skal de genetiske eller fysisk-modifikationer, der skal udføres uden at påvirke den virus native struktur og virus-funktion. Majs rayado fino virus (MRFV) kappeproteiner selv samle i Escherichia coli, der producerer stabile og tomme VLP&#39;er, der er stabiliseret med protein-protein interaktioner og som kan anvendes i virus-baserede teknologier applikationer 8. VLP&#39;er produceret i tobaksplanter blev undersøgt som et stillads, på hvilken forskellige peptider kan være kovalent vises 13. Her beskriver vi trinene til 1) bestemme, hvilken af ​​de opløsningsmiddel-tilgængelige cysteiner i en virus capsid fås til modifikation, og 2) Bioconjugate peptider til de modificerede capsider. Ved anvendelse af native eller mutation-indsat aminosyrerester og standard koblingsmidler teknologi, har en bred vifte af materialer er vist på overfladen af plantevirus, såsom Brome mosaikvirus 3, Carnation mottle virus 12, Cowpea chlorotisk skjoldet virussatellit 6, tobak mosaik virus 17, Turnip yellow mosaic virus 1 og MRFV 13.

Forbigående ekspression af mutant MRFV coat protein (CP) gener i N. benthamiana planter i en PVX-baserede vektor producerende VLP&#39;er er beskrevet i figur 1.. Den modificerede MRFV kappeproteingen er amplificeret ved PCR og derefter placeret under transkriptionel kontrol af den duplikerede subgenomiske CP promotoren i en PVX-baseret vektor, pP2C2S 2 (en gave fra D. Baulcombe, Sainsbury Laboratories, Norwich, England). I vitro RNA-transkription producerer RNA-transkripter...

Watch this Scientific Journal Video about Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

En fremgangsmåde til at analysere opløsningsmidlet adgang til thiolgruppen af ​​cysteinrester af<em&gt; Majs rayado fino virus</em&gt; (MRFV)-viruslignende partikler (VLP&#39;er) efterfulgt af et peptid tværbindingsreaktion er beskrevet. Fremgangsmåden drager fordel af tilgængeligheden af ​​adskillige kemiske grupper på overfladen af ​​VLP&#39;er, der kan være mål for specifikke reaktioner.

Analyse af opløsningsmidlet Tilgængelighed af cysteinrester på<em&gt; Majs rayado fino virus</em&gt; Virus-lignende partikler produceret i<em&gt; Nicotiana benthamiana</em&gt; Planter og tværbinding af peptider til VLP&#39;er

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

Research

JoVE Journal

Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Isolering og genom analyse af enkelte Virioner benytter &quot;enkelt virus Genomics &#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analyse af epitelbeskadigelse Produceret af<em&gt; Entamoeba histolytica</em&gt; Infektion

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In vitro analyse af MyD88-medieret Cellular immunrespons til West Nile Virus mutantstamme Infektion

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Modeling Lifecycle af Ebola virus Under biosikkerhedsniveau 2 Betingelser med virus-lignende partikler indeholdende Tetracistronic minigenomer

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrofil Isolering og analyse for at fastslå deres rolle i lymfom Cell Følsomhed til terapeutiske midler

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Udvikling af Stem Cell-afledte antigen-specifikke regulatoriske T celler mod Autoimmunitet

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

Anvendelse af Soft-agar overlay teknik til at screene for bakterielt produceret inhibitoriske forbindelser

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

Ved at kombinere analysen af DNA i en rå Virion ekstraktion med analysen af RNA fra inficerede blade til at opdage nye Virus genomer

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

Hurtig, sikker og simpel manuel sengelamper nukleinsyre udvinding til påvisning af Virus i blodprøver

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Etablering af Viral infektion og analyse af Host-Virus interaktion i Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...