Name: chemoselective modification of viral surfaces via bioorthogonal click chemistry
Uploaded: 2012-08-19T14:00:00
Description: Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

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Se tabel 1 for fremstilling af alle medier, buffere og løsninger henvises til i denne protokol. 1. Produktion af Aha-mærket Adenovirus <ol><li> Fremstille elleve 100 mm vævskulturskåle af humane embryonale nyreceller (HEK 293) opretholdes i HEK 293 cellevækstmedium (se tabel 1) ved 37 ° C, indtil de har nået 80 - 90% konfluens. (Bemærk:. Kulturer med konfluens over 90% kan være vanskeligt at inficere og kan ikke producere virus som ønsket) </li><...

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S., Lindholm, L., Boulanger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+Type+5+Fiber+Knob+Domain+has+a+Critical+Role+in+Fiber+Protein+Synthesis+and+Encapsidation.">Adenovirus Type 5 Fiber Knob Domain has a Critical Role in Fiber Protein Synthesis and Encapsidation.</a> J. Gen. Virol. 87, 3151-3160 (2006).</li><li>Zhang, M. M., Tsou, L. K., Charron, G., Raghavan, A. S., Hang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+fluorescence+imaging+of+dynamic+S-acylation+and+protein+turnover.">Tandem fluorescence imaging of dynamic S-acylation and protein turnover.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 8627-8632 (2010).</li><li>Banerjee, P. S., Ostapchuk, P., Hearing, P., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unnatural+amino+acid+incorporation+onto+adenoviral+(Ad)+coat+proteins+facilitates+chemoselective+modification+and+retargeting+of+Ad+type+5+vectors.">Unnatural amino acid incorporation onto adenoviral (Ad) coat proteins facilitates chemoselective modification and retargeting of Ad type 5 vectors.</a> J. Virol. 85, 7546-7554 (2011).</li><li>Hong, V., Presolski, S. I., Ma, C., 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=Analysis+and+Optimization+of+Copper-Catalyzed+Azide-Alkyne+Cycloaddition+for+Bioconjugation.">Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation.</a> Angew. Chem. Int. Ed. 48, 9879-9883 (2009).</li><li>Best, M. 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Udviklingen af ​​kemoselektive og bioorthogonal klik reaktioner, herunder azider, er en hastigt udviklende område af forskning, og efterfølgende er der et stigende antal af disse reaktioner for at vælge imellem for bioconjugation applikationer. Vi har begrænset rækkevidden af ​​denne protokol til kun at omfatte to metoder, der blev valgt på grund af deres anvendelighed i vores eget laboratorium og kommercielle tilgængelighed af alle reagenser. I den første vej - stamme-fremme...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> Adenovirus type 5 (Ad5) indeholdende en GFP transgen </td><td> BCBC </td><td> 391 </td></tr><tr><td> Human embryonisk nyre (HEK 293-celler) </td><td> ATCC </td><td> CRL-1573 </td></tr><tr><td> Dulbeccos Modified Eagle Medium (DMEM), High Glucose </td><td> Invitrogen </td><td> 11965-092 </td></tr><tr><td> Dulbeccos Modified Eagle Medium, ingen methionin, intet cystein (DMEM-Met /-Cys), High Glucose </td><td> Invitrogen </td><td> 21013-024 </td></tr><tr><td> Bovint kalveserum </td><td> Invitrogen </td><td> 16170-078 </td></tr><tr><td> Penicillin - streptomycin 100 × Solution (Pen Strep) </td><td> Invitrogen </td><td> 15140-122 </td></tr><tr><td> 0,5% trypsin-EDTA (10 x) </td><td&gt; Invitrogen </td><td> 15400-054 </td></tr><tr><td> 100 mm celledyrkningsskål, vævs-kultur behandlet polystyren </td><td> BD Falcon </td><td> 353003 </td></tr><tr><td> Cellekultur CO2 inkubator </td><td></td><td></td></tr><tr><td> Hæmocytometer </td><td></td><td></td></tr><tr><td> Biosikkerhed kabinet </td><td></td><td></td></tr><tr><td> Sterilt sprøjtefilter (0,2 um, celluloseacetat) </td><td> VWR </td><td> 28145-477 (NA), 514-0061 (Europa) </td></tr><tr><td> Avanti JE centrifuge </td><td> Beckman Coulter </td><td> 369001 </td></tr><tr><td> Koniske rør (50 ml sterilt) </td><td> BD Falcon </td><td> 352098 </td></tr><tr><td> Tube, Thinwall, Ultra-Clear, 13,2 ml, 14 x 89 mm </td><td> Beckman </td><td> 344059 </td></tr><tr><td> Ultracentrifuge udstyret med en SW 41 og SW 60 rotor </td><td> Beckman </td><td></td></tr><tr><td> Eppendorf Biopur Safe-Lock rør, 1,5 ml </td><td> Eppendorf </td><td> 0030 121.589 </td></tr><tr><td> Centrifuge 5418 </td><td> Eppendorf </td><td> 5418 </td></tr><tr><td> Centri-Sep gelfiltreringsmetoder spinkolonner </td><td> Princeton Separationer </td><td> CS-901 </td></tr><tr><td> Steril nål, 18 gauge </td><td></td><td></td></tr><tr><td> Kvælstof handskeposen (hvis deoxygenated CuAAC skal udføres) </td><td></td><td></td></tr><tr><td> Dewarkolbe </td><td></td><td></td></tr><tr><td> Flydende nitrogen </td><td></td><td></td></tr><tr><td> Elektroforesecelle </td><td></td><td></td></tr><tr><td> Fluorescerende gel scanner </td><td></td><td></td></tr><tr><td> Ready Gel Tris-HCI Gel </td><td> Bio-Rad </td><td> 161-1105 </td></tr><tr><td> L-Azidohomoalanine </td><td> Hjælp af Anaspec </td><td> 63669 </td></tr><tr><td> Jena Biosciences </td><td> CLK-AA005 </td><td></td></tr><tr><td> Peracetylerede N-azidoacetylgalactosamine (Ac 4 GalNAz) </td><td> Invitrogen </td><td> C33365 </td></tr><tr><td> Thermo Scientific </td><td> 88905 </td><td></td></tr><tr><td> Sigma-Aldrich </td><td> A7480 </td><td></td></tr><tr><td> Bathophenanthroline disulfonsyre (BDA) dinatriumsalt </td><td> MP Biomedicals </td><td> 0215011201 </td></tr><tr><td> Dimethylsulfoxid (DMSO) </td><td></td><td></td></tr><tr><td> Methanol </td><td></td><td></td></tr><tr><td> Tris-base (tris (hydroxymethyl) aminomethan) </td><td></td><td></td></tr><tr><td> Dinatriumphosphat (Na2 HPO 4) </td><td></td><td></td></tr><tr><td> Phosphatbufret saltvand (PBS) </td><td></td><td></td></tr><tr><td> 1M HCI </td><td></td><td></td></tr><tr><td> Glycerol </td><td></td><td></td></tr><tr><td> Bovint Serum Albumin </td><td></td><td></td></tr><tr><td> L-cystein </td><td></td><td></td></tr><tr><td> L-methionin </td><td></td><td></td></tr><tr><td> SDS (natriumdodecylsulfat) </td><td></td><td></td></tr><tr><td> 2-mercaptoethanol </td><td></td><td></td></tr><tr><td> Glycin </td><td></td><td></td></tr><tr><td> Bromphenolblåt </td><td></td><td></td></tr><tr><td> Cæsiumchlorid (CsCI) </td><td></td><td> &amp; Nbsp; </td></tr><tr><td> Kobber (I) bromid (CuBr) </td><td></td><td></td></tr><tr><td> Calciumchlorid (CaCl2) </td><td></td><td></td></tr><tr><td> Kaliumchlorid (KCl) </td><td></td><td></td></tr><tr><td> Magnesiumchlorid (MgCl2) </td><td></td><td></td></tr><tr><td> Natriumchlorid (NaCl) </td><td></td><td></td></tr><tr><td colspan="3"></td></tr><tr><td> Alkyn probe til CuAAC * </td><td></td><td></td></tr><tr><td> TAMRA Alkyn </td><td> Invitrogen </td><td> T10183 </td></tr><tr><td colspan="3"></td></tr><tr><td> Belastede alkyn probe til SPAAC * </td><td></td><td></td></tr><tr><td> TAMRA DIBO Alkyn </td><td> Invitrogen </td><td> C10410 </td></tr></tbody></table> * Bemærkelsesværdige leverandører af klik kemi reagenser og kits omfatter Invitrogen, Jena Biosciences, Berry Associates, Sigma-Aldrich, Glen Research, klik kemi Funktioner, og Baseclick. En række af alkyn farvestoffer og målrettet ligander kan findes i disse leverandørers kataloger.

chemoselective modification of viral surfaces via bioorthogonal click chemistry

Ændringen af viruspartikler har modtaget en betydelig opmærksomhed for sit enorme potentiale for at påvirke genterapi, onkolytiske applikationer og udvikling af vacciner. 1,2,3 nuværende tilgange til at ændre virale overflader, som for det meste genetik-baserede, ofte lider af dæmpning af virus produktion, infektivitet og celletransduktion. 4,5 Brug kemoselektive klik kemi, har vi udviklet en simpel alternativ metode, som forbigås disse spørgsmål, mens de resterende både meget fleksibel og tilgængelig. 1,2 Målet med denne protokol er at demonstrere effektiviteten af ​​at anvende bioorthogonal klik kemi at modificere overfladen af ​​adenovirus type 5-partikler. Denne to-trins proces, kan anvendes både terapeutisk 1 eller analytisk, 2,6, da det tillader kemoselektiv ligering af målsøgningsmolekyler, farvestoffer eller andre molekyler af interesse på proteinerforudmærket med azid tags. De tre vigtigste fordele ved denne metode er, at (1) metabolisk mærkning viser ringe eller ingen indvirkning på vira, 1,7 (2) en bred vifte af effektor ligander kan anvendes, og (3) er det bemærkelsesværdigt hurtigt, pålideligt og nemt at få adgang. 1,2,7 I det første trin af denne procedure er adenoviruspartikler fremstilles under enten azidohomoalanine (Aha, en methionin surrogat) eller unaturlige sukker O-bundet N-azidoacetylglucosamine (O-GlcNAz), som begge indeholder azidet (-N3) funktionel gruppe. Efter oprensning af azid-modificerede viruspartikler er en alkyn probe indeholdende fluorescerende TAMRA del ligeres i en kemoselektiv måde til præ-mærkede proteiner eller glycoproteiner. Endelig er en SDS-PAGE-analyse udført for at demonstrere den vellykkede ligering af proben på de virale capsidproteiner. Aha inkorporering er vist at mærke alle virale capsidproteiner (Hexon, Penton og Fiber), mens O-GlcNAz inkorporering resulterer i mærkning af Fiber alene. I denne udvikling område er flere metoder til azid-alkyn ligering med held blevet udviklet, men kun de to vi har fundet at være mest hensigtsmæssigt demonstreres heri - stamme-fremmet azid-alkyn cycloaddition (SPAAC) og kobber-katalyseret azid-alkyn cycloaddition (CuAAC) under afiltet atmosfære.

Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Adenoviruspartikler er manipuleret til at indeholde enten naturlig aminosyre analog azidohomoalanine eller azido sukker<em&gt; O</em&gt;-GlcNAz. Af azidgruppen af ​​hver er kemoselektivt ligeret via &quot;klik&quot;-kemiske reaktioner som et middel til viral overflademodifikation.

Kemoselektiv Modifikation af virale overflader via Bioorthogonal klik kemi

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

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