Low-cost COVID-19 vaccine could improve herd immunity around the world NatureComms
hosts. In addition to DCFHP, the constructs were designed to express glutamine synthetase. Sequence confirmation of the DCFHP gene and whole plasmid sequence was routinely conducted. In addition, Leap-In Transposase system ORF expression constructs were designed and constructed based on ATUM’s proprietary technology.hosts containing DCFHP genes were maxi-prepped .
. Resulting pooled cells were sorted and resulting single clones were analyzed as described throughout the manuscript. Optimal clones were selected for cell banking.SARS-CoV-2 VOCs and SARS-CoV-1 spike pseudotyped lentiviral particles were produced. HEK293T cells were transfected with plasmids described above for pseudoviral production using BioT transfection reagent. Six million cells were seeded in D10 media in 10-cm plates 1 day before transfection.
A virus dilution was made containing the virus of interest and D10 media . Virus dilutions into media were selected such that a suitable signal would be obtained in the virus-only wells. Polybrene was present at a final concentration of 5 µg/mL in all samples. 50 µL of heat inactivated sera was mixed with 50 µL viral dilution to make a final volume of 120 µl In each well.
The inhibitor and virus mixture was left to incubate for 1 h at 37 °C. After incubation, the medium was removed from the cells on the plates made 1 day prior. This was replaced with 100 µl of inhibitor/virus dilutions and incubated at 37 °C for approximately 48 h. Infectivity readout was performed by measuring luciferase levels.
. LOQ was set as the neutralizing titer of day 0 serum or the lowest serum dilution tested, whichever was higher. For samples with different LOQs on the same graph, the average value was used.RBD was plated in 50 µl in each well on a MaxiSorp microtiter plate in 1xPBS and left to incubate for at least 1 h at room temperature.
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