Objective To develop high-immunogenicity antigens targeting the BA.5 variant of SARS-CoV-2 and to screen for monoclonal antibodies with high neutralizing and blocking activity,providing new strategies for the development of vaccines and diagnostic reagents.Methods The ZP protein gene fragment from zebrafish vitel-logenin and the HIS protein tag were inserted into the pCDNA3.4 plasmid to construct the recombinant plasmid pCDNA3.4-RBD(BA.5)-ZP-HIS,which was then transfected into 293F cells to express the RBD-ZP protein.The expression of the protein was verified by SDS-PAGE and its binding capabilities to ACE2 receptor molecules and aluminum adjuvant were detected.The immunogenicity of the fusion protein was evaluated using a BALB/c mouse model,and monoclonal antibodies were prepared through hybridoma technology.Monoclonal antibodies with strong neutralizing and blocking activity were screened and their neutralizing activity was detected by blocking ELISA.Results The ZP gene and HIS protein tag sequence were successfully inserted into the pCDNA3.4 vector and the RBD-ZP protein with a molecular weight of 50 kDa was successfully expressed.The immunogenicity test results showed that the ZP protein effectively enhanced the immunogenicity of the RBD protein and improved its binding capability to the ACE2 receptor.After immunizing mice with the RBD-ZP protein,8 monoclonal antibodies that specifically bind to both the mutant and wild-type strains were cloned and screened through hybridoma technology,among which 3 could effectively block the binding of the SARS-CoV-2 RBD protein to the human ACE2 receptor.Conclusion This study successfully expressed the RBD-ZP fusion protein,which significantly enhanced the immunogenicity and receptor binding capability of the RBD protein.Three monoclonal antibodies with high neutral-izing and blocking activity were screened out,providing strong support for the development of COVID-19 vaccines and diagnostic reagents..

Objective To develop high-immunogenicity antigens targeting the BA.5 variant of SARS-CoV-2 and to screen for monoclonal antibodies with high neutralizing and blocking activity,providing new strategies for the development of vaccines and diagnostic reagents.Methods The ZP protein gene fragment from zebrafish vitel-logenin and the HIS protein tag were inserted into the pCDNA3.4 plasmid to construct the recombinant plasmid pCDNA3.4-RBD(BA.5)-ZP-HIS,which was then transfected into 293F cells to express the RBD-ZP protein.The expression of the protein was verified by SDS-PAGE and its binding capabilities to ACE2 receptor molecules and aluminum adjuvant were detected.The immunogenicity of the fusion protein was evaluated using a BALB/c mouse model,and monoclonal antibodies were prepared through hybridoma technology.Monoclonal antibodies with strong neutralizing and blocking activity were screened and their neutralizing activity was detected by blocking ELISA.Results The ZP gene and HIS protein tag sequence were successfully inserted into the pCDNA3.4 vector and the RBD-ZP protein with a molecular weight of 50 kDa was successfully expressed.The immunogenicity test results showed that the ZP protein effectively enhanced the immunogenicity of the RBD protein and improved its binding capability to the ACE2 receptor.After immunizing mice with the RBD-ZP protein,8 monoclonal antibodies that specifically bind to both the mutant and wild-type strains were cloned and screened through hybridoma technology,among which 3 could effectively block the binding of the SARS-CoV-2 RBD protein to the human ACE2 receptor.Conclusion This study successfully expressed the RBD-ZP fusion protein,which significantly enhanced the immunogenicity and receptor binding capability of the RBD protein.Three monoclonal antibodies with high neutral-izing and blocking activity were screened out,providing strong support for the development of COVID-19 vaccines and diagnostic reagents..