Effect of N-glycosylation Modification of Recombinant SARS-CoV-2 Receptor-binding Domain Vaccine Candidate on Immunogenicity
Objective:Protein glycosylation plays an important role in regulating the immunogenicity of vaccines.The receptor-binding domain(RBD)of the spike protein of the novel coronavirus(SARS-CoV-2)contains two N-glycosylation sites(N331 and N343).This study aims to investigate the effect of RBD glycosylation on protein expression levels and immunogenicity.Methods:Wild-type RBD antigen and three N-glycosylation site mutations of RBD antigens,including wild-type RBD-WT and deglycosylated antigens(RBD-N1,RBD-N2,and RBD-2N),were constructed and expressed using glycosylation engineering yeast and mammalian cell expression systems.The purified antigens were obtained and the antibody levels induced by recombinant RBD vaccines were compared in immunized mice.Results:RBD glycosylation site mutations significantly affected protein expression levels,with similar characteristics in yeast and mammalian cell expression systems.Mutations at any N-glycosylation site resulted in decreased expression levels,especially when the N343 glycosylation site was mutated or both sites were mutated simultaneously.The results of RBD antigen immunization in mice showed that the mutation of the N343 glycosylation site resulted in a significant decrease in the titers of specific antibodies and neutralizing antibodies.Conclusions:Glycosylation modification is critical for the expression level and immunogenicity of the RBD protein,with the N343 glycosylation site playing a critical role in regulating protein expression level and immunogenicity.
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