Construction,screening and immunogenicity of the recombinant poxvirus vaccine rVTT△TK-RBD against SARS-CoV-2
Objective To construct a recombinant poxvirus vector vaccine,rVTT△TK-RBD,and to evaluate its safety and immunogenicity.Methods The receptor-binding domain(RBD)gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE,to construct the recombinant poxvirus shuttle vector pSTKE-RBD.This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain(VTT).The recombinant poxvirus rVTT△TK-RBD was successfully obtained after several rounds of fluorescence phage screening.The effect of rVTT△TK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination.The levels of specific and neutralizing antibodies produced by rVTT△TK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly.The effect of rVTT△TK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry.Results Through homologous recombination,enhanced green fluorescent protein(EGFP)screening marker,and multiple rounds of fluorescent phosphorescence phage screening,a recombinant poxvirus rVTT△TK-RBD,expressing RBD with deletions in the thymidine kinase(TK)gene,was successfully obtained,which was validated by PCR.The in vivo experiments on BALB/c mice showed that rVTT△TK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT.Conclusion The recombinant poxvirus vaccine rVTT △TK-RBD against SARS-CoV-2 is successfully constructed and obtained,with its safety and immunogenicity confirmed through various experiments.
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