Construction and Identification of A Non-Replicating Recombinant Adenovirus Expressing the S1 and N Proteins of SARS-CoV-2
We aimed to construct a replication-deficient recombinant adenovirus vaccine fused with the S1 antigen and N antigen of severe acute respiratory syndrome coronavirus(SARS-CoV-2),and provide a basis for further research on the immunogenicity of the vaccine.The recombinant adenovirus plasmid pKAd5-S1-N inserted into the S1-N gene of SARS-CoV-2 was constructed by overlap polymerase chain reaction and homologous recombination.The recombinant plasmid was transfected into HEK293A cells to obtain the virus species AdV5-S1-N and amplified.The virus was purified by density-gradient centrifugation using cesium chloride.The virus titer was measured by limited dilution.Western blotting was used to verify expression of the Sl-N fusion protein of the AdV5-S1-N recombinant adenovirus.Immunization of BALB/c mice with AdV5-S1-N vaccine was through intramuscular injection.On day 14,we collected blood from the submandibular vein,and measured the specific antibody titers of serum against the antigens of S1 and N proteins using ELIS As.Titers with 9.45 × 1011 IU/mL of AdV5-Sl-N were obtained.Western blotting showed that,after infection of AdV5-S1-N cells,the fusion protein could be expressed normally and bind specifically to anti-S1 protein and anti-N protein antibodies.ELISAs showed that mice immunized with recombinant adenovirus could produce specific IgG antibodies targeting S1 and N proteins.A replication-deficient adenovirus vector was used to obtain a high-titer recombinant adenovirus that fused and expressed the S1-N protein of the SARS-CoV-2 Omicron mutant strain.The recombinant virus could induce specific immune responses against S1 and N proteins in mice,laying the foundation for further research and evaluation of this candidate vaccine.
Severe acute respiratory syndrome coronavirus-2Adenovirus-based vector vaccineHumoral immunityFusion expression
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