Two alternative strategies were explored to remove contaminating luciferase proteins from virus stocks. Progeny virions in culture supernatants (IAV stocks) or released through one freeze/thaw cycle from infected cells (RSV stocks) were cleared (4,000×g for 20 minutes at 4°C), then pelleted (60,000×g for 30 minutes at 4°C). Pelleted material was resuspended in TNE buffer (50 mM Tris/Cl pH 7.2, 10 mM EDTA) and purified through a 20/60% one-step sucrose gradient in TNE buffer (100,000×g for 90 minutes at 4°C). Virions were harvested from the gradient intersection. Alternatively, cleared RSV stocks were purified and polished through size exclusion and binding chromatography by passaging through dual functionality Capto Core 700 resin (GE Healthcare) using an ÄKTA avant chromatography system (GE Healthcare). After purification through either method, virus stocks were stored in aliquots at −80°C.
Capto core 700 resin
Manufactured by GE Healthcare
Capto Core 700 resin is a chromatography resin designed for purification of biomolecules. It features a porous agarose bead structure and is suitable for use in packed bed chromatography columns.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 1000, by Thermo Fisher Scientific (4 mentions)
Pyrophosphatase enzymes, by Aldevron (2 mentions)
Ribogreen assay, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Aldevron (2 mentions)
Rnase inhibitor, by Aldevron (2 mentions)
S adenosyl methionine, by Aldevron (2 mentions)
Kta avant chromatography system, by GE Healthcare (1 mentions)
Akta purifier 100, by GE Healthcare (1 mentions)
Guanylyl transferase, by Aldevron (1 mentions)
T7 polymerase, by Aldevron (1 mentions)
Rntp mix, by New England Biolabs (1 mentions)
5 protocols using capto core 700 resin
1
Purifying Viral Particles from Luciferase
2
Purification and Characterization of H5N8 and H5N1 Vaccine Candidates
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Vaccine candidate viruses were inoculated into embryonated chicken eggs (10–11 days old) and incubated at 34 °C for 60 h. After incubation, the allantoic fluid was harvested and centrifugated at 1800× g for 30 min. Then, the virus solution was inactivated with 0.01% formaldehyde and concentrated five times via cross-flow ultrafiltration with a 500 kDa membrane cassette. The inactivated virus was further purified with Capto Core 700 resin (GE Healthcare) in AKTA Purifier 100 system. Finally, the purified bulk was filtered with a 0.22 μm Sartobran P filter and stored at 4 °C.
A/Vietnam/1194/2004 NIBRG-14(H5N1) virus obtained from NIBSC was also inoculated into embryonated chicken eggs, and then the allantoic fluid was harvested and centrifugated. The following procedures of the H5N1 vaccine preparation were the same as the H5N8 vaccine.
For quantitation of haemagglutinin in the vaccine candidate virus, the virus concentrates of H5N8 and H5N1 viruses were deglycosylated using PNGase F and analyzed by SDS-PAGE. The amount of HA as a percentage of total protein was then calculated by dividing the ‘total HA’ by the ‘total protein’ [19 ]. The total proteins of the purified virus concentrates were assayed using a BCA protein assay.
A/Vietnam/1194/2004 NIBRG-14(H5N1) virus obtained from NIBSC was also inoculated into embryonated chicken eggs, and then the allantoic fluid was harvested and centrifugated. The following procedures of the H5N1 vaccine preparation were the same as the H5N8 vaccine.
For quantitation of haemagglutinin in the vaccine candidate virus, the virus concentrates of H5N8 and H5N1 viruses were deglycosylated using PNGase F and analyzed by SDS-PAGE. The amount of HA as a percentage of total protein was then calculated by dividing the ‘total HA’ by the ‘total protein’ [19 ]. The total proteins of the purified virus concentrates were assayed using a BCA protein assay.
3
Generation of Stabilized mRNA Stocks
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Generation of saRNA stocks was achieved by T7 promoter-mediated in vitro transcription using NotI-linearized DNA template. In vitro transcription was performed using an in-house-optimized protocol using T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes procured from Aldevron. DNA plasmid was digested away (DNase I, Aldevron) and cap0 structures were added to the transcripts by vaccinia capping enzyme, GTP, and S-adenosyl-methionine (Aldevron). RNA was then purified from the transcription and capping reaction components by chromatography using a CaptoCore 700 resin (GE Healthcare) followed by diafiltration and concentration using tangential flow filtration. The saRNA material was terminally filtered with a 0.22 μm polyethersulfone filter and stored at −80°C until use. All saRNA was characterized by agarose gel electrophoresis and quantified both by UV absorbance (NanoDrop 1000) and Ribogreen assay (Thermo Fisher). OVA-expressing mRNA was obtained from a commercial vendor (TriLink CleanCap OVA mRNA, L-7610).
4
YFV(wt) VLP Production and Purification
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The cDNA sequence encoding the wild-type prM-E protein sequence of YFV “Angola71” strain (Genbank #AY968064) was codon optimized, synthetized, and cloned into plasmid VRC8400 (VRC/NIAID/NIH, Bethesda, MD, USA) at Genscript (Piscataway, NJ, USA) for expression of YFV(wt) VLPs. The signal peptide used was the wild-type signal peptide from “Angola71” YFV strain. Transient transfection of HEK293F cells, harvest and clarification were carried out as described for ZIKV VLPs. Yellow fever VLPs were purified by liquid chromatography in 50 mM Tris pH 8.5 buffer, using Sartobind Q membrane adsorber (Sartorius) and Captocore 700 resin (GE Healthcare), as previously described (45 (link), 46 (link)).
5
In Vitro Transcription of saRNA
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Vaccine saRNA was generated by T7 polymerase-mediated in vitro transcription (IVT) using NotIlinearized DNA plasmids as templates. An in-house optimized IVT protocol was used with commercially available rNTP mix (NEB) and commercially available T7 polymerase, RNase inhibitor, and pyrophosphatase enzymes (Aldevron, Fargo, ND). DNA plasmids were digested away (DNase I, Aldevron), and Cap 0 structures were added to the transcripts by treatment with guanylyltransferase (Aldevron), GTP, and S-adenosylmethionine (NEB). RNA was chromatographically purified using Capto Core 700 resin (GE Healthcare, Chicago, IL) followed by diafiltration and concentration through tangential flow filtration using a 750 kDa molecular weight cut-off (MWCO) modified polyethersulfone (mPES) membrane (Repligen, Waltham, MA). Terminal filtration of the saRNA material was done using a 0.22 µm PES filter, and the saRNA materials were stored at -80°C until use/complexation. Agarose gel electrophoresis was used to characterize saRNA size and integrity, and RNA concentration was quantified by UV absorbance (NanoDrop 1000) and RiboGreen assay (Thermo Fisher Scientific, Waltham, MA).
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