Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection.
The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoter-based vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and assayed for the immunoprecipitation of either p120 or p80 protein from cytopathic or non-cytopathic BVDV biotype-infected bovine cells. The p80 gene sequence was also integrated into a baculovirus genome for its expression in Spodoptera frugiperda insect cells. The recombinant proteins isolated from bacteria or insect cells showed distinct antigenic properties when analysed by ELISA. Their ability to detect anti-BVDV specific antibodies was examined in a monoclonal antibody-based competitive ELISA performed on a series of field cattle sera. This comparative assay revealed the superiority of the insect cell-mediated expression to mimic the natural BVDV antigen produced by cell culture. The baculovirus/insect cell recombinant antigen gave the highest correlation between the ELISA-detected antibodies and the corresponding virus neutralization data.
Eurogentec s.a., Parc de recherches de la Cense Rouge, Seraing, France.
N
N
Vanderheijden
L
L
De Moerlooze
D
D
Vandenbergh
G
G
Chappuis
A
A
Renard
C
C
Lecomte
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli; D008970:Molecular Weight; D009036:Moths; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins