For stimulation of ex vivo cell samples, OLP peptide pools of 15-mers with 11 amino acid overlap were generated spanning the SARS-CoV-2 Spike RBD (R319-S591, GenScript). Sequences that contained VOC mutations were exchangeable with the corresponding mutated peptides due to a modular OLP pool design.
Sars cov 2 spike rbd
Manufactured by GenScript
Sourced in United States, China
The SARS-CoV-2 Spike RBD is a recombinant protein that represents the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. The RBD is responsible for the virus's attachment to the human ACE2 receptor, which is the first step in the viral infection process. This product can be used for research purposes, such as in the development of diagnostic tests, vaccines, and therapeutics targeting the SARS-CoV-2 virus.
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3 protocols using sars cov 2 spike rbd
1
SARS-CoV-2 Spike RBD Peptide Stimulation
2
SARS-CoV-2 Spike RBD Aptamer Sensor
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The materials used in this study were 3-aminopropyl triethoxysilane (APTES) (Sigma Aldrich); aptamer DNA sequence was based on Song et al. (2020) – [C6–NH2]–CAG CAC CGA CCT TGT GCT TTG GGA GTG CTG GTC CAA GGG CGT TAA TGG ACA that was synthesized by Bioneer Corporation. Bovine serum albumin (BSA), cerium nitrate hydrate (Ce[NO3])3·6H2O), glutaraldehyde 50%, phosphate buffer saline (PBS) pH 7.4, and solid potassium ferricyanide K3[Fe(CN)6] (Sigma Aldrich); potassium chloride (KCl) (Merck), sodium hydroxide (NaOH) (Merck, pa); SARS-CoV-2 Spike RBD (GenScript, USA), double distilled water (PT. Ikapharmindo Putramas Indonesia), and inactivated avian virus (H5N1) was a gift from PT. Tekad Mandiri Citra, Bandung, Indonesia. Clinical samples of COVID-19 patients used in this study were obtained from the Covid-19 test laboratory (C-29 Lab.) at Padjadjaran University.
3
Yeast-Expressed SARS-CoV-2 RBD Variants
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The SARS-CoV-2 Spike RBD (S protein 318-531, GenBank: YP_009724390) encoding gene was synthesized by GenScript (Nanjing, Jiangsu, China) and subcloned into BamHI endonuclease sites of pYD1 plasmid. The variants B.1.1.7 (N501Y), B.1.351 (N501Y/E484K/K417N), and B.1.617.1 (L452R/E484Q) were constructed by a site-directed mutagenesis kit (see Section 2.7) with temple of wild-type RBD. The S. cerevisiae EBY200 strain was constructed by deleting the gal80 gene of S. cerevisiae EBY100 strain. The resultant shuttle plasmid pYD1-RBD was transformed into S. cerevisiae EBY200. Recombinant yeast transformants were plated on selective minimal dextrose plates at 30°C for 3 days. The positive clones were confirmed by DNA sequencing and then induced in YNB-CAA-Glucose medium (0.67% YNB, 0.5 casamino acids, 2% glucose) at 30°C for 24 h for RBD expression. Meanwhile, S. cerevisiae EBY200 containing empty pYD1 plasmid was used as a negative control for the following tests.
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