Ecl reagent

SARS-CoV-2 RBD dodecamer mRNA was transfected into HEK293T cells by Lipofectamine™ 2000 according to the manufacturer's instructions (Thermo Fisher Scientific, 11668027, Waltham, MA, USA). Supernatants and cell lysates were obtained, and the expression of SARS-CoV-2 RBD dodecamer antigen in vivo was detected by SARS-CoV-2 RBD protein detection ELISA kit (Vazyme, 7E510D1, Nanjing, China) and Western blotting (WB). Briefly, cells were lysed with Cell Lysis Reagent (Sigma–Aldrich, C3228-50 ML, St. Louis, MO, USA) containing protease inhibitor (Thermo Fisher Scientific, 78,430, Waltham, MA, USA), denatured at 100 °C for 10 min, separated by electrophoresis on 10% SDS-PAGE gels, and then transferred to nitrocellulose transfer membranes (GE Amersham Biosciences, 10-6000-01, Pittsburgh, PA). Membranes were incubated with RBD primary Abs overnight (Proteintech Europe, 67758-1-Ig, Deansgate, UK), and then incubated with corresponding secondary Abs conjugated to HRP (Santa Cruz Biotechnology, sc-2005, Dallas, TX, USA). Finally, the relative expression levels of protein were detected using ECL reagents (Santa Cruz Biotechnology, sc-2048, Dallas, TX, USA) and quantified by Quantity One software [Bio-Rad Laboratories, Quantity One®1-D, Hercules, CA, USA)].