Cloning and expression of bovine coronavirus genes

The nucleocapsid (N) protein and the two subunits of spike protein (S1 and S2) of bovine coronavirus (BCV), were individually expressed in Spodopetra frugiperda (Sf9) insect cells using recombinant baculovirus vectors. BCV RNA was extracted from MDBK infected cells and the coding sequence of each BCV target gene was amplified using RT-PCR. The RT-PCR products were cloned into the baculovirus shuttle vector; pBlueBac4.5/V5-His TOPO and the recombinant plasmids that carry target genes in correct orientation were identified. The cloned genes were introduced in 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). Recombinant baculoviruses were selected by plaque purification; verified for the presence of target genes using PCR and propagated for generation of high-titer viral stock. Infection of insect cells with the recombinant baculoviruses revealed high-level expression of the target proteins as indicated by immunofluoresent test, solid phase ELISA, and Western blot.