Objective To express the receptor binding domain(RBD)protein of severe acute respiratory symptom coronavi-rus 2(SARS-CoV-2)in soluble form by prokaryotic system and evaluate its immunogenicity by immunizing mice with the purified protein,so as to provide a new idea for the preparation of SARS-CoV-2 candidate vaccine.Methods SARS-CoV-2 RBD gene fragment was amplified and ligated with pNCMO2 vector after enzyme digestion.The obtained recombinant pro-karyotic expression plasmid pNC-RBD was transformed into Bacillus choshinensis competent cells,which were subjected to expanded culture and induced by lowering temperature.The target protein was purified by affinity chromatography,adsorbed by aluminum hydroxide adjuvant,and then the female BALB/c mice were immunized subcutaneously in the back using the protein,10 mice for each group,with the aluminum adjuvant-free protein as control.The humoral immunity was measured by ELISA,while the cellular immune effect by proliferation assay in vitro.The secretion of various cytokines in vitro was measured by ELISA,and the cell typing was determined by flow cytometry.Results The recombinant expression plasmid pNC-RBD was constructed correctly as identified by colony PCR and sequencing.The recombinant protein had a relative molecular mass of about 22 000,which could be expressed and secreted,and the purity of recombinant protein after purifi-cation was over 95％.The titers of binding antibody and neutralizing antibody in serum of the immunized mice were about 1∶10 000 and 1∶750,respectively,which were significantly higher than those in adjuvant group(t=2.845 and 2.528,respectively,each P＜0.01).The recombinant protein stimulated lymphocyte proliferation and promoted the secretion of IL-1β and I FNγ in vitro.The results of cell typing test showed that the lymphocyte proliferation of both CD4+and CD8+T cells was promoted.Conclusion The RBD protein of SARS-CoV-2 was successfully expressed in soluble form with good immuno-genicity,which lays a foundation for the development of SARS-CoV-2 genetic engineering vaccine based on RBD protein.
Severe acute respiratory symptom coronavirus 2(SARS-CoV-2)Receptor binding domain(RBD)proteinSoluble protein expressionImmunogenicity
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