Name: a "plug-and-display" nanoparticle vaccine platform based on outer membrane vesicles displaying sars-cov-2 receptor-binding domain
Uploaded: 2022-07-25T14:30:00
Description: Watch this Scientific Journal Video about A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain at JoVE.com

Dette arbejde blev støttet af Key Program of Chongqing Natural Science Foundation (nr. cstc2020jcyj-zdxmX0027) og Chinese National Natural Science Foundation Project (nr. 31670936, 82041045).

1. Plasmid konstruktion
<ol><li>Indsæt DNA-kodning af SpyCatcher-sekvens (supplerende fil 1) i et ampicillinresistent pThioHisA-ClyA-plasmid (se Materialetabel) mellem BamH I- og Sal I-stederne for at konstruere plasmidet pThioHisA ClyA-SC efter en tidligere offentliggjort rapport20.</li>
	<li>Sammensæt det syntetiserede SpyTag-RBD-Histag fusionsgen (supplerende fil 1) i et pcDNA3.1 plasmid (se Materialetabel<...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+vesicle+vaccines.">Outer membrane vesicle vaccines.</a> Seminars in Immunology. 50, 101433 (2020).</li><li>Toyofuku, M., Nomura, N., Eberl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+and+origins+of+bacterial+membrane+vesicles.">Types and origins of bacterial membrane vesicles.</a> Nature Reviews Microbiology. 17 (1), 13-24 (2019).</li><li>Sartorio, M. G., Pardue, E. J., Feldman, M. F., Haurat, M. 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Q., Peng, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+outer+membrane+vesicles+as+potential+biological+nanomaterials+for+antibacterial+therapy.">Bacterial outer membrane vesicles as potential biological nanomaterials for antibacterial therapy.</a> Acta Biomaterialia. 140, 102-115 (2022).</li><li>Hoffmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+cell+entry+depends+on+ACE2+and+TMPRSS2+and+is+blocked+by+a+clinically+proven+protease+inhibitor.">SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.</a> Cell. 181 (2), 271-280 (2020).</li><li>Robbiani, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+antibody+responses+to+SARS-CoV-2+in+convalescent+individuals.">Convergent antibody responses to SARS-CoV-2 in convalescent individuals.</a> Nature. 584 (7821), 437-442 (2020).</li><li>Wang, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2:+Structure,+Biology,+and+Structure-Based+Therapeutics+Development.">SARS-CoV-2: Structure, Biology, and Structure-Based Therapeutics Development.</a> Frontiers in Cellular and Infection Microbiology. 10, 587269 (2020).</li><li>Yang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunogenicity+of+a+recombinant+tandem-repeat+dimeric+RBD-based+protein+subunit+vaccine+(ZF2001)+against+COVID-19+in+adults:+Two+randomised,+double-blind,+placebo-controlled,+phase+1+and+2+trials.">Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD-based protein subunit vaccine (ZF2001) against COVID-19 in adults: Two randomised, double-blind, placebo-controlled, phase 1 and 2 trials.</a> The Lancet Infectious Disease. 21 (8), 1107-1119 (2021).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+vaccine-elicited+antibodies+to+SARS-CoV-2+and+circulating+variants.">mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.</a> Nature. 592 (7855), 616-622 (2021).</li><li>Amanat, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARS-CoV-2+mRNA+vaccination+induces+functionally+diverse+antibodies+to+NTD,+RBD,+and+S2.">SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD, and S2.</a> Cell. 184 (15), 3936-3948 (2021).</li><li>Tan, H. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+of+prime-boost+protein+subunit+vaccine+strategies+against+SARS-CoV-2+in+mice+and+macaques.">Immunogenicity of prime-boost protein subunit vaccine strategies against SARS-CoV-2 in mice and macaques.</a> Nature Communication. 12 (1), 1403 (2021).</li><li>Thapa, H. B., Mueller, A. M., Camilli, A., Schild, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intranasal+vaccine+based+on+outer+membrane+vesicles+against+SARS-CoV-2.">An intranasal vaccine based on outer membrane vesicles against SARS-CoV-2.</a> Frontiers in Microbiology. 12, 752739 (2021).</li><li>Ma, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+vaccines+based+on+the+receptor+binding+domain+(RBD)+and+heptad+repeat+(HR)+of+SARS-CoV-2+elicit+robust+protective+immune+responses.">Nanoparticle vaccines based on the receptor binding domain (RBD) and heptad repeat (HR) of SARS-CoV-2 elicit robust protective immune responses.</a> Immunity. 53 (6), 1315-1330 (2020).</li><li>Yang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBD-modified+bacterial+vesicles+elicited+potential+protective+immunity+against+SARS-CoV-2.">RBD-modified bacterial vesicles elicited potential protective immunity against SARS-CoV-2.</a> Nano Letters. 21 (14), 5920-5930 (2021).</li><li>Rhinesmith, T., Killinger, B. A., Sharma, A., Moszczynska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimer-PAGE:+A+method+for+capturing+and+resolving+protein+complexes+in+biological+samples.">Multimer-PAGE: A method for capturing and resolving protein complexes in biological samples.</a> Journal of Visualized Experiments. (123), e55341 (2017).</li><li>Arslan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+total+protein+and+bioactive+protein+concentrations+in+bovine+colostrum.">Determining total protein and bioactive protein concentrations in bovine colostrum.</a> Journal of Visualized Experiments. (178), e63001 (2021).</li><li>Alves, N. J., Turner, K. B., Walper, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+protein+packaging+within+outer+membrane+vesicles+from+Escherichia+coli:+Design,+production+and+purification.">Directed protein packaging within outer membrane vesicles from Escherichia coli: Design, production and purification.</a> Journal of Visualized Experiments. (117), e54458 (2016).</li><li>Kim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+transcriptomic+landscape+of+Escherichia+coli+BL21(DE3).">Genomic and transcriptomic landscape of Escherichia coli BL21(DE3).</a> Nucleic Acids Research. 45 (9), 5285-5293 (2017).</li><li>Daleke-Schermerhorn, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoration+of+outer+membrane+vesicles+with+multiple+antigens+by+using+an+autotransporter+approach.">Decoration of outer membrane vesicles with multiple antigens by using an autotransporter approach.</a> Applied and Environmental Microbiology. 80 (18), 5854-5865 (2014).</li><li>Kuipers, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+outer+membrane+vesicles+displaying+high+densities+of+pneumococcal+antigen+at+the+surface+offer+protection+against+colonization.">Salmonella outer membrane vesicles displaying high densities of pneumococcal antigen at the surface offer protection against colonization.</a> Vaccine. 33 (17), 2022-2029 (2015).</li><li>Veggiani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+polyproteams+built+using+twin+peptide+superglues.">Programmable polyproteams built using twin peptide superglues.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (5), 1202-1207 (2016).</li><li>Saunders, K. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+antibody+vaccine+for+pandemic+and+pre-emergent+coronaviruses.">Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.</a> Nature. 594, 553-559 (2021).</li><li>van Saparoea, H. B. V., Houben, D., Kuijl, C., Luirink, J., Jong, W. S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+protein+ligation+systems+to+expand+the+functionality+of+semi-synthetic+outer+membrane+vesicle+nanoparticles.">Combining protein ligation systems to expand the functionality of semi-synthetic outer membrane vesicle nanoparticles.</a> Frontiers in Microbiology. 11, 890 (2020).</li><li>Needham, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+the+innate+immune+response+by+combinatorial+engineering+of+endotoxin.">Modulating the innate immune response by combinatorial engineering of endotoxin.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (4), 1464-1469 (2013).</li><li>Zanella, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome-minimized+outer+membrane+vesicles+from+Escherichia+coli+as+a+generalized+vaccine+platform.">Proteome-minimized outer membrane vesicles from Escherichia coli as a generalized vaccine platform.</a> Journal of Extracellular Vesicles. 10 (4), 12066 (2021).</li><li>Wang, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncating+the+structure+of+lipopolysaccharide+in+Escherichia+coli+can+effectively+improve+poly-3-hydroxybutyrate+production.">Truncating the structure of lipopolysaccharide in Escherichia coli can effectively improve poly-3-hydroxybutyrate production.</a> ACS Synthetic Biology. 9 (5), 1201-1215 (2020).</li><li>Liu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+vesicles+from+flagellin-deficient+Salmonella+enterica+serovar+Typhimurium+induce+cross-reactive+immunity+and+provide+cross-protection+against+heterologous+Salmonella+challenge.">Outer membrane vesicles from flagellin-deficient Salmonella enterica serovar Typhimurium induce cross-reactive immunity and provide cross-protection against heterologous Salmonella challenge.</a> Scientific Reports. 6, 34776 (2016).</li><li>Balhuizen, M. D., Veldhuizen, E. J. A., Haagsman, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane+vesicle+induction+and+isolation+for+vaccine+development.">Outer membrane vesicle induction and isolation for vaccine development.</a> Frontiers in Microbiology. 12, 629090 (2021).</li><li>Hua, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+immunomodulator+delivery+platform+based+on+bacterial+biomimetic+vesicles+for+enhanced+antitumor+immunity.">A novel immunomodulator delivery platform based on bacterial biomimetic vesicles for enhanced antitumor immunity.</a> Advanced Materials. 33 (43), 2103923 (2021).</li></ol>

For at skabe en "Plug-and-Display" nanopartikelvaccineplatform blev SC-fusioneret ClyA udtrykt i BL21 (DE3) stammer, som er en af de mest anvendte modeller til rekombinant proteinproduktion på grund af dens fordele i proteinekspression24, så der ville være nok SC-fragment, der vises på overfladen af OMV'erne under processen med bakterieproliferation. Samtidig blev der udarbejdet et ST-smeltet målantigen til den kemiske kobling mellem antigenerne og OMV'erne. Denne forsøgsordnings fordele og ...

Som en potentiel vaccineplatform har ydre membranvesikler (OMV'er) tiltrukket sig mere og mere opmærksomhed i de senere år 1,2. OMV'er, der hovedsageligt udskilles naturligt af gramnegative bakterier3, er sfæriske nanoskalapartikler sammensat af et lipiddobbeltlag, normalt i størrelsen 20-300 nm4. OMV'er indeholder forskellige forældrebakteriekomponenter, herunder bakterielle antigener og patogenassocierede molekylære mønstre (PAMP'er), der tjener som faste immunpotentiatorer5. Ved at drage fordel af deres unikke komponenter, naturlige vesikelstruktur og store genteknologiske modifikationssteder er OMV'er blevet udviklet til brug inden for mange biomedicinske områder, herunder bakterielle vacciner6, adjuvanser7, kræftimmunterapilægemidler8, lægemiddelafgivelsesvektorer9 og antibakterielle klæbemidler10.
SARS-CoV-2-pandemien, der har spredt sig over hele verden siden 2020, har taget hårdt på det globale samfund. Det receptorbindende domæne (RBD) i spike-protein (S-protein) kan binde med humant angiotensin-konverterende enzym 2 (ACE2), som derefter medierer virusets indtræden i cellen11,12,13. RBD ser således ud til at være et primært mål for vaccineopdagelse14,15,16. Imidlertid er monomer RBD dårligt immunogen, og dens lille molekylvægt gør det vanskeligt for immunsystemet at genkende, så adjuvanser kræves ofte17.
For at øge immunogeniciteten af RBD blev OMV'er, der viste polyvalente RBD'er, konstrueret. Eksisterende undersøgelser, der bruger OMV til at vise RBD, smelter normalt RBD sammen med OMV, der skal udtrykkes i bakterier18. RBD er imidlertid et virusafledt protein, og prokaryot ekspression vil sandsynligvis påvirke dets aktivitet. For at løse dette problem blev SpyTag (ST) / SpyCatcher (SC) -systemet, der stammer fra Streptococcus pyogenes, brugt til at danne et kovalent isopeptid med OMV og RBD i et konventionelt buffersystem19. SC-domænet blev udtrykt med Cytolysin A (ClyA) som et fusionsprotein af bioengineered Escherichia coli, og ST blev udtrykt med RBD via HEK293F cellulært ekspressionssystem. OMV-SC og RBD-ST blev blandet og inkuberet natten over. Efter oprensning ved ultracentrifugering eller størrelsesekskluderende kromatografi (SEC) blev omv-rbd opnået.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ampicillin sodium</td>
			<td>Sangon Biotech</td>
			<td>A610028</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>Countstar</td>
			<td>BioTech</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein quantification Kit</td>
			<td>cwbio</td>
			<td>cw0014s</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Touching Imaging System</td>
			<td>Bio-rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Danamic Light Scattering</td>
			<td>Malvern</td>
			<td>Zetasizer Nano S90</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>Cavoy</td>
			<td>Power BV</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ-Buffers H 10X TBST Buffer</td>
			<td>Sangon Biotech</td>
			<td>C520009</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat pAb to mouse IgG1</td>
			<td>Abcam</td>
			<td>ab97240</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed freezing centrifuge</td>
			<td>Bioridge</td>
			<td>H2500R</td>
			<td></td>
		</tr>
		<tr>
			<td>His-Tag mouse mAb</td>
			<td>Cell signaling technology</td>
			<td>2366s</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sangon Biotech</td>
			<td>A600277</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl beta-D-thiogalactopyranoside</td>
			<td>Sangon Biotech</td>
			<td>A600118</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA His-Bind Superflow</td>
			<td>Qiagen</td>
			<td>70691</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powdered milk</td>
			<td>Sangon Biotech</td>
			<td>A600669</td>
			<td></td>
		</tr>
		<tr>
			<td>OPM-293 cell culture medium</td>
			<td>Opm biosciences</td>
			<td>81075-001</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1 RBD-ST plasmid</td>
			<td>Wuhan genecreat biological techenology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>ZSGB-bio</td>
			<td>ZLI-9061</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine Linear</td>
			<td>Polysciences</td>
			<td>23966-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Prestained protein ladder</td>
			<td>Thermo</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>pThioHisA ClyA-SC plasmid</td>
			<td>Wuhan genecreat biological techenology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF Western Blotting Membranes</td>
			<td>Roche</td>
			<td>03010040001</td>
			<td></td>
		</tr>
		<tr>
			<td>Quixstand benchtop systems (100 kD hollow fiber column)</td>
			<td>GE healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE loading buffer (5x)</td>
			<td>Beyotime</td>
			<td>P0015</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>A100241</td>
			<td></td>
		</tr>
		<tr>
			<td>Supersignal west pico PLUS (enhanced chemiluminescence solution)</td>
			<td>Thermo</td>
			<td>34577</td>
			<td></td>
		</tr>
		<tr>
			<td>Suspension instrument</td>
			<td>Life Technology</td>
			<td>Hula Mixer</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Hitachi</td>
			<td>HT7800</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid</td>
			<td>LP0042B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman coulter</td>
			<td>XPN-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraviolet spectrophotometer</td>
			<td>Hitachi</td>
			<td>U-3900</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sangon Biotech</td>
			<td>A610961</td>
			<td></td>
		</tr>
	</tbody>
</table>

a "plug-and-display" nanoparticle vaccine platform based on outer membrane vesicles displaying sars-cov-2 receptor-binding domain

Biomimetiske nanopartikler opnået fra bakterier eller vira har tiltrukket sig betydelig interesse for vaccineforskning og -udvikling. Ydre membranvesikler (OMV'er) udskilles hovedsageligt af gramnegative bakterier under gennemsnitlig vækst med en diameter i nanostørrelse og selvadjuverende aktivitet, hvilket kan være ideelt til vaccinelevering. OMV'er har fungeret som et mangesidet leveringssystem for proteiner, nukleinsyrer og små molekyler. For at drage fuld fordel af OMV'ernes biologiske egenskaber blev bioengineerede Escherichia coli-afledte OMV'er anvendt som bærer og SARS-CoV-2-receptorbindende domæne (RBD) som et antigen til at konstruere en "Plug-and-Display" vaccineplatform. SpyCatcher (SC) og SpyTag (ST) domænerne i Streptococcus pyogenes blev anvendt til konjugering af OMV'er og RBD. Cytolysin A (ClyA) genet blev oversat med SC-genet som et fusionsprotein efter plasmidtransfektion, hvilket efterlod et reaktivt sted på overfladen af OMV'erne. Efter blanding af RBD-ST i et konventionelt buffersystem natten over blev der dannet kovalent binding mellem OMV'erne og RBD. Således blev der opnået en multivalent OMV-vaccine. Ved at erstatte med forskellige antigener kan OMV-vaccineplatformen effektivt vise en række heterogene antigener og derved potentielt hurtigt forhindre infektionssygdomsepidemier. Denne protokol beskriver en præcis metode til konstruktion af OMV-vaccineplatformen, herunder produktion, oprensning, biokonjugation og karakterisering.

Rutediagrammet for denne protokol er vist i figur 1. Denne protokol kan være en generel tilgang til at bruge OMV'er som en vaccineplatform; man behøver kun at vælge de passende ekspressionssystemer baseret på typen af antigener.
Figur 2 giver et gennemførligt plasmiddesignskema. SC-genet er forbundet med ClyA-genet via en fleksibel linker, mens ST forbinder til 5'-terminalen af RBD-genet med et His-tag-gen til oprensning og verif...

Watch this Scientific Journal Video about A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain at JoVE.com

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Den nuværende protokol beskriver bioengineering af ydre membranvesikler som en "Plug-and-Display" vaccineplatform, herunder produktion, oprensning, biokonjugation og karakterisering.

En "plug-and-display" nanopartikelvaccineplatform baseret på ydre membranvesikler, der viser SARS-CoV-2-receptorbindende domæne

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane ...

Nanovidenskab kan effektivt forbedre det antigenspecifikke immunrespons efter at have vist antigener på overfladen af Den største fordel ved denne vaccineplatform kan opnå go off-plak og display. Med andre ord er platformen i stand til at vise de mange forskellige antigener i Denne metode kan også anvendes på andre biologiske nanopartikler til at vise motor til nano-størrelse bærere. For at udføre cytolysin En spionkatetertransformation skal du starte med at tilføje fem mikroliter plasmaopløsning til 50 mikroliter BL 21 kompetent stamme.Blæs forsigtigt og lad afkøle på is i 30 minutter. Anbring opløsningen i 90 sekunder i vandbadet ved 42 grader Celsius, og sæt derefter straks den blandede opløsning på is i tre minutter. Tilsæt 500 mikroliter luria bertani medium til bakteriesuspensionen og efter blanding af kultur ved 220 rotationer i minuttet ved 37 grader Celsius i en time.Dernæst plade al transformation luria bertani agar plade indeholdende ampicillin og kultur natten over ved 37 grader Celsius. For OMV spycatcher produktion, start med at isolere en enkelt koloni fra pladen til 20 ml luria bertani medium og kultur natten over ved 220 rotationer i minuttet ved 37 grader Celsius. Inkuber bakterieopløsningen i et to liters medium og kultur ved 220 rotationer i minuttet ved 37 grader Celsius i fem timer, indtil det logaritmiske vækststadium er nået.Tilsæt isopropyl beta D-IPTG for at gøre den endelige koncentration af bakterieopløsningen til at være 0,5 millimolar og derefter kultur natten over ved 220 rotationer pr. Minut ved 25 grader Celsius. Udfør derefter OMV spycatcher rensning. Start med centrifusing af bakterieopløsningen.Filtrer supernatanten med et 0,22 mikrometer membranfilter, og koncentrer det derefter ved hjælp af en hul fibersøjle. Filtrer koncentratet gennem et 0,22 mikrometer membranfilter, centrifuger det derefter ved 150.000 gange G ved fire grader Celsius i to timer ved hjælp af en ultracentrisikring, og kassér supernatanten med en pipette. Suspender nedbøren igen med PBS, og opbevar den ved minus 80 grader Celsius.For at udføre RBD spytag-transfektion skal du begynde med at vælge et passende eukaryot ekspressionssystem og dyrke cellerne natten over ved 130 rotationer i minuttet ved 37 grader Celsius efter genopretning. Tilsæt 20 mikroliter HEK 293F celleopløsning i den automatiserede celletæller. Optag antallet af celler, juster koncentrationen til en gang 10 til de seks celler pr. Milliliter.Og derefter dyrke cellerne ved 130 rotationer i minuttet ved 37 grader Celsius i fire timer. Filtrer RBD spy tag plasmid gennem et 0,22 mikrometer membranfilter og tilsæt 300 mikrogram plasmider i cellekulturmediet, indtil det endelige volumen er 10 ml. Ryst i 10 sekunder.Varm PEI til 65 grader Celsius i vandbadet. Bland 0,7 ml PEI med 9,3 ml cellekulturmedium, og ryst intermitterende i 10 sekunder. Tilsæt plasmidopløsning til PEI-opløsningen.Ryst blandingen intermitterende i 10 sekunder, og inkuber den ved 37 grader Celsius i 15 minutter. Tilsæt blandingen til 280 ml cellekulturmedium og kultur ved 130 rotationer pr. Minut ved 37 grader Celsius i fem dage. Udfør derefter RBD spytag rensning.Start med centrifusing af cellerne ved 6.000 gange G ved 25 grader Celsius i 20 minutter, og brug en pipette til at opsamle supernatanten. Fyld søjlen med to milliliter nikkel NTA agarose og vask den tre gange med PBS. Tilsæt imidazol i cellesupernatanten for at gøre den endelige koncentration på 20 millimolar, og læg cellesupernatanten.Der tilsættes tre kolonnevolumener PBS indeholdende 20 millimolar imidazol til vask, og vaskefraktionen opsamles. Gradientelue med tre kolonnevolumener PBS indeholdende lave til høje koncentrationer af imidazolelut to gange for hver koncentration. For at bestemme proteinkoncentrationen ved BCA-metoden fortyndes standard BSA-proteinopløsningen serielt fra to til 0,0625 milligram pr. milliliter, fortyndes den oprensede OMV-spionfanger og RBD-spionmærke 10 gange.Bland derefter BCA-arbejdsløsningerne A og B i forholdet 50 til én. Tilsæt fortyndet proteinopløsning, og bland med BCA-arbejdsopløsning. Inkuberes ved 37 grader i 30 minutter.Absorbansen måles ved 562 nanometer af hver brønd, og proteinkoncentrationen beregnes ud fra standardkurven. For at udføre biokonjugation af OMV spycatcher og RBD spy tag, bland OMV spycatcher og RBD spytag og PBS i et forhold på 40 til en. Drej lodret for at blande blandingen natten over ved 15 rotationer i minuttet ved fire grader Celsius.Udfør rensning af OMV RBD ved hjælp af PBS-opløsning ved hjælp af en procedure svarende til OMV spycatcher oprensning. Plasmadesignskemaet viser, at spionfangergenet er forbundet med cly A-genet via fleksibel linker, mens spytag forbinder til de fem primære terminaler af RBD-genet med et hys-tag-gen til oprensning og verifikation. Efter ultracentrifugering forblev næsten alle de ikke-reagerede RBD-spytag i supernatanten.Den anden ultracentrifugering gav ikke en yderligere signifikant fordel i forhold til første gang. Den gennemsnitlige hydrodynamiske diameter af OMV spycatcher var 133 nanometer, mens den var 152,6 nanometer for OMV-RBD. Disse forskelle kan skyldes, at RBD øger OMV-partikelstørrelsen efter intensiv visning.Resultaterne af TEM er i overensstemmelse med partikelstørrelsesfordelingen opnået fra DLS-resultaterne. Rotation til blanding af blandingen er meget vigtig. Reaktionens effektivitet kan være meget lavere uden rotation.

Research

JoVE Journal

Bioengineering

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Hud tatovering som en ny tilgang til DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Stretching Micropatterned Cells on a PDMS Membrane

Strækker micropatterned Celler på en PDMS Membrane

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Papir-baserede enheder til isolering og karakterisering af ekstracellulær Vesikler

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

Fremstilling af en funktionaliseret Magnetic Bakteriel Nanocellulose med Iron Oxide Nanopartikler

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

Målrettet plasmamembran Levering af en hydrofob Cargo indkapslet i en Liquid Crystal Nanopartikel Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

Tør Film Photoresist-baserede elektrokemisk mikrofluid Biosensor Platform: Enhed fabrikation, On-chip Assay forberedelse og drift

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

Lab-on-a-CD-platform til generering af flercellede tredimensionelle Sfæroider

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Elektrovåd-baserede Digital Microfluidics Platform for Automatiseret Enzym-forbundet Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

En Lab-On-A-Chip platform til stimulering af Osteocyte Mechanotransduction og analyse af funktionelle resultater af Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

Generering af en forenklet tredimensionel skin-on-a-chip-model i en mikromaskiner med mikrofluidisk platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...