Construction of a Recombinant Infectious Clone of HCoV-OC43
Objective:To construct an infectious clone of HCoV-OC43 carrying the green fluorescent protein reporter gene.Methods:The whole genome sequence of the HCoV-OC43 infectious clone carrying the green fluorescent protein reporter gene was designed and synthesized into 32 segments.The segments were then assembled into 8 fragments by fusion PCR.Recombinant plasmids were obtained by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae.The recombinant plasmid was transfected into HEK-293T cells for virus rescue.The virus rescue was confirmed by infecting cells with the collected supernatants from transfected cells.Results:HCoV-OC43 recombinant virus carrying the green fluorescent protein was successfully rescued from infectious clone transfected cells.Conclusions:HCoV-OC43 infectious clone carrying the green fluorescent protein can be obtained using transfor-mation-associated recombination cloning method.The infectious clone provides an effective tool in basic and applied research on human coronaviruses.
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