HIGH-LEVEL EXPRESSION OF THE ENVELOPE GLYCOPROTEIN (GP53) OF BOVINE VIRAL DIARRHEA VIRUS (SINGER) AND ITS POTENTIAL USE AS DIAGNOSTIC REAGENT

A 1.74-kb cDNA fragment containing the gp53 coding region has been clo ned from bovine viral diarrhoea virus (BVDV) strain Singer by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis in dicated that gp53 of BVDV strains Singer, NADL and SD-1 shared extensi ve sequence homology at both the RNA (85-94%) and protein (82-91%) lev els. Nineteen cysteine residues and five potential N-linked glycosylat ion sites were identified within the sequenced region, all of which we re conserved. These observations suggest that although the homology at the nucleotide sequence level may vary, there was strong structural c onservation among bovine viral diarrhoea virus envelope proteins. Full -length gp53 was expressed in Escherichia coli as a fusion protein wit h glutathione-S-transferase (GST). The N-terminal half of gp53 was als o synthesised in E. coli as a 28-KDa recombinant protein using the T7 RNA polymerase-directed expression system. Both recombinant proteins w ere expressed at high levels (approximately 30-50 mg/l). The recombina nt proteins were recognised in ELISA and Western blot analyses by poly clonal serum raised against a mixture of BVDV and classical swine feve r virus (CSFV). Rabbit antiserum raised against the 28-kDa recombinant protein reacted with different BVDV strains in ELISA and immunofluore scent antibody test, but not with CSFV in the same tests. These result s demonstrated that the bacterial recombinant proteins have similar im munological properties to that of the native viral protein and, in con junction with its homologous antisera, can be useful as diagnostic rea gents.