pSG3Δenv  plasmid BioVector NTCC质粒载体菌株细胞基因保藏中心

Name名称：BioVector® pSG3Δenv 
Description描述：Human 293T and HeLa cells were obtained from the American Type Culture Collection and grown in Dulbecco modified Eagle medium (ThermoFisher) supplemented with 10% fetal calf serum (Atlanta Biologicals) at 37C in 5% CO2. Cell cultures were confirmed to be negative for mycoplasma in routine tests (Lonza) every six months. Viral particles were produced by Calcium Phosphate transfection (Takara) of 293T cells with the pSG3Δenv plasmid, obtained from the NIH AIDS Reagent Program (Catalog # 11051). Virions produced from this construct lack the viral Env protein, but are fully infectious when pseudotyped with envelope glycoproteins from HIV-1 or VSV ((13-15) and confirmed for our construct). For virion production, 2.5×106 293T cells were seeded on 10 cm plates and transfected 24 h later. 18 h post transfection, media was removed and replaced with fresh media. 48 h post transfection, virion-containing media was removed and filtered through a 0.45 μM membrane, layered on a 20% sucrose cushion in HS buffer (10 mM Hepes pH 7.4 and 140 mM NaCl), and centrifuged for 2 h at 28,000 rpm in a SW32 Ti rotor (Beckman). After centrifugation, media was decanted and virion-containing pellets were resuspended in 100 μl HS buffer per 10 cm plate of transfected cells, aliquoted, flash frozen in liquid nitrogen, and stored at −80C. Virion CA concentrations were determined by HIV-1 p24 ELISA assay (XpressBio). Typical yields were 40-100 pmol CA per 10 cm plate. HeLa cell lysates were prepared by seeding 2.8×106 cells on a 15 cm plate. After 48 h, media was removed and cells were washed with PBS, removed from the plate with a cell lifter (Sigma Aldrich), placed in a micro-centrifuge tube, and centrifuged for 1 min at 1500 rpm (4C). After centrifugation, excess PBS was removed and cells were resuspended in 200 μl of lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 50 μg/ml melittin) per 15 cm plate and incubated on ice for 15 min. Lysates were then spun in a micro-centrifuge for 10 min at 17,000 rpm. The soluble fraction was collected, aliquoted, flash frozen in liquid nitrogen, and stored at −80C.
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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