Expression of the bovine coronavirus spike glycoprotein subunits in insect cells using recombinant baculoviruses

In the present study, the bovine coronavirus (BCV) spike glycoprotein cleavage products (S1 and S2) were individually expressed in Spodoptera frugiperda (Sf9) insect cells, using a baculovirus expression system. The coding sequence of S1 and S2 gene fragments were amplified by RT-PCR and cloned into the baculovirus shuttle vector pBlueBac4.5/V5-His TOPO ® TA. The cloned fragments were inserted into the genome of Autographa californica nuclear polyhydrosis virus (AcMNPV) under control of the polyhedrin promoter, through a process of homologous recombination between the shuttle vector and a linearized replication-defective baculovirus DNA (Bac-N-BlueTM). Recombinant baculoviruses were selected by plaque purification; verified for the presence of target sequences using PCR and propagated for generation of high-titer viral stocks. Infection of insect cells with the recombinant baculoviruses revealed high-level expression of the target proteins as indicated by immunofluoresent test and solid phase ELISA using BCV-specific polyclonal antiserum.