Name: expression and purification of virus-like particles for vaccination
Uploaded: 2016-06-02T15:00:00
Description: Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

Vi ønsker at anerkende de forudgående medlemmer af Ross lab, der har hjulpet optimere protokollen for maksimal effektivitet og udbytter.

1. pattedyrsekspression System for Generation of Influenza H3N2 VLP <ol><li> Subklon viral strukturelle glycoproteiner hæmagglutinin (HA), neuraminidase (NA) og humant immundefektvirus (HIV) Gag p55 i eukaryote ekspressionsvektorer, såsom tidligere beskrevne pTR600. 7 </li><li> Amplificere DNA i kemisk kompetente Escherichia coli (fx DH5a) og isolere transfektion-grade plasmid under anvendelse af et plasmid-oprensningskit ifølge producentens instruktioner. Mængden af ​​DNA er afhængig af u...

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Vi har anvendt de ovenfor beskrevne teknikker med succes udtrykke og oprense SVPs og VLP&#39;er sammensat af flere strukturelle proteiner til forskellige patogener. Generelt anvender vi pattedyrekspressionssystemer at generere vores VLP&#39;er. i vores hænder, mammal-celleafledt imidlertid Chik VLP udbytter var lave. Chik VLP udbytte var mere robust, når du bruger et rekombinant baculovirus-insekt celle system. Generelt baculovirus-insektcellesystemer giver højere mængder af rekombinante proteiner, som kan skyldes d...

Viruslignende partikler (VLP&#39;er) er en godkendt teknologi til vaccination af mennesker. Faktisk er nogle af de mere contemporarily licenserede vacciner, herunder det humane papillomavirus (HPV) og hepatitis Β (HepΒ) vacciner anvender denne fremgangsmåde. VLP&#39;er er dannet af strukturelle proteiner stand til selv-samling. De samlede partikler efterligne virale morfologier, men kan ikke inficere eller replikere fordi de mangler virale genomer. VLP&#39;er kan udtrykkes og oprenses fra en række prokaryote og eukaryote systemer. En gennemgang af litteraturen viste, at forskellige ekspressionssystemer er ansat på følgende satser: bakterier - 28%, gær - 20%, plante - 9%, insekt - 28%, og pattedyr - 15% 1. Af note, er HPV-vacciner baseret på L1-capsidprotein produceret i gær (Gardasil) eller i en insektcelle-system (Cervarix) 2. HepΒ vacciner, Recombivax og Engerix-Β, fremstilles også i gær, og er sammensat af HepΒ overfladeantigen 34. Vi anvender pattedyr og insekter-ekspressionssystemer til frembringelse af VLP&#39;er der kræver co-ekspression af multiple strukturelle proteiner til samling. Vores arbejde fokuserer på at designe, producere, og rensningsprocesser VLP-baserede vacciner mod humane patogener: influenzavirus, respiratorisk syncytialvirus (RSV), dengue virus (DENV), og chikungunya virus (CHIKV). Vores metoder er fleksible nok til at muliggøre co-ekspression af de multiple strukturelle proteiner fra flere ekspressionsplasmider, eller et enkelt ekspressionsplasmid (figur 1). Vi har tidligere fremstillet og oprenset H5N1 VLP&#39;er samlet af co-ekspression af plasmider, der koder influenza hemagglutinin (HA), neuraminidase (NA) og matrix (M1) i humane embryonale nyre 293T-celler 5,6. Generne blev kodon-optimeret til ekspression i mammale celler og klonet ind pTR600, en eukaryot ekspressionsvektor indeholdende cytomegalovirus immediate-early-promotor plus intron A til initiering transcription af eukaryote indsatser og bovint væksthormon polyadenyleringssignalet for terminering af transskription 7. En lignende fremgangsmåde under anvendelse af tre-plasmid co-ekspression af HA, NA (figur 1A) og et alternativt viral matrix protein, HIV Gag p55, blev anvendt til generering af sæsoninfluenza subtype human H3N2 VLP&#39;er i denne undersøgelse og er blevet vist at frembringe VLP&#39;er af lignende størrelse som influenza partikler 8. Selvom influenzavacciner overvejende fremkalder anti-HA-antistoffer, tilsætning af influenza-neuraminidase medierer sialidase aktivitet at aktivere VLP spirende fra transficerede celler 9 og også præsentere yderligere immunogene mål. For at producere RSV VLP&#39;er, vi valgte også den uafhængige kerne af HIV Gag at designe prototypiske vacciner, der præsenterer udelukkende RSV overfladeglycoproteiner for yderligere at vise fleksibilitet VLP-dannelse ved hjælp af HIV Gag, som tidligere beskrevet og karakteriseret ved elektronmikroskopi 6,10. Andrehar tidligere vist, at VLP&#39;er præsenterer RSV-glycoproteiner kan samles ved anvendelse af forskellige virale komponenter fra Newcastle Disease-virus (NDV) 11, og influenza-matrix 12. Fuld længde overfladeglycoprotein sekvenser blev brugt i dette studie at fastholde native konformationer, som kan være nødvendige for funktionel receptorbinding og antistofgenkendelse assay gennem enzymkoblet immunosorbentassay (ELISA). Vores eksempler på brug af enkelte plasmid ekspressionssystemer til at generere partikler er DENV og CHIKV. I tilfælde af DENV, kan vi producere subvirale partikler (SVPs) uden capsid i 293T-celler fra et enkelt plasmid indeholdende et PRM / E strukturelle gen ekspressionskassetten 13. Udtrykket SVP anvendes til at betegne den manglende en kerne eller capsidprotein ved samling af virale strukturelle proteiner. Chik VLP&#39;er kan også fremstilles under anvendelse af et enkelt plasmid indeholdende nogle CHIKV strukturelle gen kassette, der koder for capsid og kappeproteiner, eller i insect celler ved infektion med et rekombinant baculovirus der koder for det samme strukturelle gen kassette optimeret til insektcelleekspression (figur 1B - C). Slutresultatet af udtrykket tilgange beskrevet ovenfor er frigivelsen af ​​VLP&#39;er i celledyrkningsmedium, som derefter kan oprenses via ultracentrifugering gennem en 20% glycerolpude. I denne rapport præsenterer vi metoder til at udtrykke og rense disse VLP&#39;er fra pattedyr og insektcellesystemer.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Plasmid Maxi Kit</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">12163</td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM</td>
			<td style="width:167px;">Cellgro</td>
			<td>10-013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Lipofectamine</td>
			<td style="width:167px;">Life Technologies</td>
			<td>L3000075</td>
			<td>Lipofectamine 2000, Lipofectamine 3000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opti-MEM I</td>
			<td style="width:167px;">Life Technologies</td>
			<td>31985062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Bac-to-Bac Baculovirus Expression System</td>
			<td style="width:167px;">Life Technologies</td>
			<td style="width:119px;">10359-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Cimarec I 6 multipoint stirrer plate</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">50094596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Polyclear ultracentrifuge tubes</td>
			<td style="width:167px;">Seton</td>
			<td style="width:119px;">7025</td>
			<td>Confirm appropriate tubes for ultracentrifuge and bucket size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Micro BCA Protein Assay Kit</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">23235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate citrate buffer</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">P4922</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">o-phenylenediamine dihydrochloride&#160;</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">P8287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">0.22 &#956;m vacuum filter top (500 mL)</td>
			<td style="width:167px;">Nalgene</td>
			<td style="width:119px;">569-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glycerol</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">H2SO4</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:119px;">339741&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sf-900 II SFM</td>
			<td style="width:167px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10902-096</td>
			<td></td>
		</tr>
	</tbody>
</table>

expression and purification of virus-like particles for vaccination

Virus-lignende partikler (VLP&#39;er) og subvirale partikler (SVPs) er en alternativ tilgang til viral vaccine design, der giver fordelene ved øget biosikkerhed og stabilitet i forhold anvendelse af levende patogener. Ikke-infektiøse og selv-samling, der VLP bruges til at præsentere strukturelle proteiner som immunogener, uden om behovet for levende patogener eller rekombinante virale vektorer til antigen levering. I denne artikel viser vi de forskellige stadier af VLP design og udvikling til fremtidige applikationer i præklinisk dyreforsøg. Proceduren omfatter følgende faser: udvælgelse af antigen, ekspressionen af ​​antigen i cellelinje af valg, rensning af VLP / SVPs, og kvantificering for antigen dosering. Vi demonstrerer anvendelse af både pattedyr- og insektcellelinier til ekspression af vores antigener og vise, hvordan metoder adskiller sig i udbytte. Metoden præsenteres kan gælde for en række patogener og kan opnås ved at erstatte de antigener med immunogen structural proteiner af brugerens mikroorganisme af interesse. VLP&#39;er og SVPs hjælpe med antigen karakterisering og udvælgelse af de bedste vaccinekandidater.

VLP udbyttet var variabel ved virusantigen konstrukt design. I denne protokol, har vi vist brug af insekt- og mammale celler til produktion af SVP eller VLP&#39;er i supernatant og oprensning ved ultracentrifugering. Fire undertyper af DENV prM / E strukturel genekspression kassetter blev brugt til at konstruere versioner af DENV SVPs (afgrænsede som 1-4) i tabel 1 og viser et interval mellem 1,1 til 2,6 mg totalt protein i 0,6 ml volumener. For VLP, der kræver en tre ...

Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

Expression and Purification of Virus-like Particles for Vaccination

Her præsenterer vi en protokol til at syntetisere viruslignende partikler ved hjælp af enten baculovirus eller pattedyrekspressionssystemer og ultracentrifugering rensning. Denne meget tilpasselig tilgang bruges til at identificere virale antigener som vaccine mål på en sikker og fleksibel måde.

Ekspression og oprensning af viruslignende partikler til vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

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