pSARS-CoV-2 plasmid BioVector NTCC质粒载体菌株细胞基因保藏中心

Name名称：BioVector® pSARS-CoV-2
Description描述：Spike matching the sequence of the USA-WA1/2020 isolate, was generated from the CDS of pSARS-CoV-2 (NTCC® Cat# VG40589-UT) and subcloned into the NheI/XbaI linearized 5 vector pCR3.1 using Gibson assembly. To match the South African SARS-CoV2 isolate (hCoV19/South Africa/KRISP-EC-K005321) the CDS of pSARS-CoV-2 VG40589-UT was used as a template to introduce equivalent spike substitutions D80A, D215G, K417N, E484K, N501Y, D614G, A701V and deletion of the positions 242-244 with a mutation in the furin cleavage site, R683G. The substitutions and deletion were introduced by fragment amplification, overlap extension PCR, and subsequent subcloning into the NheI/XbaI linearized vector pCR3.1 using Gibson assembly. Spike and VSV-G trans-complementation for replicon delivery particle production BHK-21 cells were transfected with spike- or VSV-G-encoding plasmids using Lipofectamine 3000 (ThermoFisher #L3000001), using a reverse-transfection protocol as per manufacturer’s instructions (at 2µl reagent per ug DNA ratio). At 24h post transfection, 6 million cells were electroporated with 5µg replicon RNA and 2µg N protein mRNA as detailed above. Cells from one electroporation were seeded in a collagen-coated (Collagen from calf skin, Sigma #C8919) T75 flask or 3 electroporations were combined into a single T175 flask. For P0 imaging, some of the electroporated cells were seeded in parallel on collagen-coated 96-well plates for imaging. Supernatant was collected 24h post-electroporation for spike RDPs or 48h post-electroporation for VSV-G RDPs, and cleared by centrifugation. RDPs were concentrated via poly-ethylene glycol (PEG) precipitation, via an overnight incubation of the supernatant with a final concentration of 8% PEG6000 in 0.4M NaCl at 4°C. Subsequently, the PEG supernatant mixture was spun at 3000g for 30min. Pellets were resuspended in TNE Buffer (50mM Tris pH 7.4, 1mM EDTA, 200mM NaCl) and stored at -80°C until used.
Map图谱：
Sequence序列：
Resistance抗性：
Marker筛选标记：

Supplier生产商: BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心
http://www.biovector.net