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

ABSTRACT

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 [R] 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-Blue[TM]]. 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