![]() ![]() Antigens were captured on the chip while serial dilutions of hACE2-ECD proteins then flowed over the chip surface. d, The binding kinetics profiles of monomeric or trimeric RBD to hACE2 were assessed using a single-cycle model. c, Ultracentrifugation sedimentation profiles of RBD-trimer. The molecular in the single peak of gel filtration profile was shown in reducing SDS-PAGE. The cell supernatant was sequentially purified with Histrap excel and Superdex 200 Increase 10/300 GL column in PBS. b, Size-exclusion chromatogram and SDS-PAGE analyses of RBD-trimer. The lower panel shows the expected structures of the assembled trimeric molecule. The upper panel shows the construction of the RBD-trimer. a, Schematic diagram of the SARS-CoV-2-fusion core and SARS-CoV-2-RBD to combine as a trimeric molecule. Notably, SPR assay demonstrated that the RBD-trimer bound to hACE2 with a KD of 60.7 ± 9.5 nM, which is comparable to that of the RBD-monomer (90.3 ± 5.5 nM) ( Figure 1d), indicating that RBD-trimer accurately recovered the native conformation of SARS-CoV-2 receptor binding motifs (RBM).įigure 1 Engineering and characterizing of SARS-CoV-2 RBD-trimer. A DAS-ELISA assay demonstrated that RBD-trimer reached a high expression level of 12.1 mg/L by transient transfection, higher than that of S-ECD-trimer (4.2 mg/L), suggesting a potential of high scalable production and feasibility to meet vaccine demands worldwide (Figure S1a and c). The MW of the RBD-trimer was further corroborated as 151.1 kDa by analytical ultracentrifugation, indicating the trimeric assembly state of RBD as well ( Figure 1c). ![]() SDS-PAGE demonstrated that RBD-trimer under reducing conditions migrated at a molecular weight (MW) of ∼50 kDa, higher than expected MW (37 kDa), implying glycosylated modifications of the protein ( Figure 1b). Analytical gel filtration showed that purified RBD-trimer was eluted as a single peak with a size of ∼170 kDa ( Figure 1b), revealing the trimeric form of RBD. The supernatant of the transfected cells was collected for further sequential purification of affinity and gel filtration chromatography. RBD-trimer was transiently expressed in mammalian HEK293T cells. Alpha helix free#To avoid potential instability caused by free cysteine residue, SARS-CoV-2-RBD was truncated at K537, the position just before C538 ( Figure 1a). We designed SARS-CoV-2 RBD-trimer by connecting in tandem the HR1-linker-HR2 to SARS-CoV-2-RBD ( Figure 1a). Alpha helix plus#According to the conventional ELISA, the neutralization titers were defined as the reciprocal of the maximum dilution multiple, which according to optical absorption value at 450 nm is less than half the difference between positive and negative control value, then plus negative control value. After washing with buffer solution, the cells were incubated with rabbit anti-influenza A nucleoprotein (Abcam) for one hour. Cells were fixed with 80% acetone for 10 minutes and air-dried. The positive control was the mixture of viruses and cells, while the negative control was only cells. The mixture was subsequently transferred to the MDCK cells at a final concentration of 100 TCID 50 viruses per well and incubated for 72 hours at 37☌, 5% CO 2. Duplicate serial dilutions of heat-inactivated (30 min at 56☌) serum samples were prepared in DMEM medium without trypsin/EDTA and mixed with H1N1 A/California/07/2009 virus in assay medium containing trypsin/EDTA, for one hour at 37☌, 5% CO 2. MDCK cells were seeded in 96-well plates, at 15,000 cells per well in DMEM medium supplemented with 1% FBS. ![]() The neutralization assay was conducted in a BSL-2 facility. Then, the titers of antigens were obtained by 4-PL curves. The standard curve was fitted in a sigmoidal, 4-PL, and X is the concentration model by the exponential form of standard protein concentration and absorbance value. The plates on a microplate reader are set to 450 nm for HRP-based substrate development. Following incubation and washing, an HRP-labeled anti-human Fc antibody (Sino Biological) was added. After washing three times, 200 ng mAb GH12 protein (an RBD-specific antibody that does not compete with CB6, PDB accession number: 7D6I) was used as a secondary antibody to contact the proteins. After blocking for one hour at room temperature, 100 μL gradient concentrations of purified RBD-monomer, RBD-trimer, S-ECD-trimer proteins (from 2 μg/mL to 2 ng/mL), or 8-fold diluted supernatant to be measured were added to the plates and incubated for two hours. The Lancet Regional Health – Western PacificĢ00 ng of the CB6-Fab protein (an RBD-specific capture antibody) was coated in a microtiter plate overnight.The Lancet Regional Health – Southeast Asia.The Lancet Gastroenterology & Hepatology. ![]()
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