High level expression of the envelope glycoprotein (gp53) of bovine viral diarrhoea virus (Singer) and its potential use as diagnostic reagent.
A 1.74-kb cDNA fragment containing the gp53 coding region has been cloned from bovine viral diarrhoea virus (BVDV) strain Singer by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis indicated that gp53 of BVDV strains Singer, NADL and SD-1 shared extensive sequence homology at both the RNA (85-94%) and protein (82-91%) levels. Nineteen cysteine residues and five potential N-linked glycosylation sites were identified within the sequenced region, all of which were conserved. These observations suggest that although the homology at the nucleotide sequence level may vary, there was strong structural conservation among bovine viral diarrhoea virus envelope proteins. Full-length gp53 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). The N-terminal half of gp53 was also synthesised in E. coli as a 28-KDa recombinant protein using the T7 RNA polymerase-directed expression system. Both recombinant proteins were expressed at high levels (approximately 30-50 mg/l). The recombinant proteins were recognised in ELISA and Western blot analyses by polyclonal serum raised against a mixture of BVDV and classical swine fever virus (CSFV). Rabbit antiserum raised against the 28-kDa recombinant protein reacted with different BVDV strains in ELISA and immunofluorescent antibody test, but not with CSFV in the same tests. These results demonstrated that the bacterial recombinant proteins have similar immunological properties to that of the native viral protein and, in conjunction with its homologous antisera, can be useful as diagnostic reagents.
CSIRO Australian Animal Health Laboratory, Geelong, Victoria, Australia.
M
M
Yu
A R
AR
Gould
C J
CJ
Morrissy
H A
HA
Westbury
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis*; D002417:Cattle; D017124:Conserved Sequence; D017931:DNA Primers; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014759:Viral Envelope Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C076551:gp53, bovine viral diarrhea virus