Rescue and Identification of the Recombinant rLLR/NSP3-CoV-2/RBD Expressing SARS-CoV-2 RBD Based on LLR Backbone
The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has swept the world since December 2019.However,the immunization of children under 3 years old was absent from comprehensive strategies.Lanzhou lamb rotavirus(LLR)has been approved as a routine vaccine for the prevention of infantile Rotavirus enteritis in China.In this study,a reverse genetic technique was used to insert the receptor-binding domain(RBD)protein of SARS-CoV-2 into the ORF of LLR/NSP3 gene for recombinant virus rescue.The cytopathic effect(CPE),silver nitrate staining of dsRNA PAGE and RT-PCR amplification of NSP3 genes all verified our successful rescue of rLLR/NSP3-CoV-2/RBD recombinant virus.The NSP3 genes of the recombinant virus rLLR/NSP3-CoV-2/RBD showed obvious migration within P5 generations as expected.Furthermore,Western blot and indirect immunofluorescence had demonstrated the ability of recombinant rLLR/NSP3-CoV-2/RBD to stably express RBD of SARS-CoV-2 within P5 generations.The results of indirect immunofluorescence and qRT-PCR amplification showed that the slightly lower titer and GCEs of rLLR/NSP3-CoV-2/RBD than those of the parental rLLR at different time points did not affect the infection and replication.In this study,a reverse genetic technique was used to rescue the recombinant rLLR/NSP3-CoV-2/RBD,which could stably express the RBD protein of SARS-CoV-2 within P5 generations.This provides a new reference for further development of oral rotavirus and SARS-CoV-2 combined vaccines suitable for children under 3 years old.
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