Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection

Abstract

The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoterbased 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 pl20 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.

Human and murine antibody recognition is focused on the ATPase/Helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein

“…3 The serine-protease domain (p80) of BVDV p125 has been shown to induce strong humoral responses, which correlate with virus neutralization in both vaccinated and infected animals. 33,34 Recently, it was proposed that antibodies to HCV NS3 are mostly conformation-dependent, because only lowlevel reactivities have been detected using NS3 peptides as solid-phase ligands. 22,23 This is one reason why the fine specificity of B-cell recognition of the domains of NS3 are not as well characterized as compared with other HCV proteins containing multiple linear B-cell determinants.…”

Characterization of the immune response of cattle against non-cytopathic and cytopathic biotypes of bovine viral diarrhoea virus.

“…Construction and characterization of recombinant BVDV Osloss strain NS3 protein were recently described (Vanderheijden et al, 1993). Briefly, the NS3 gene isolated from NcoI-BamHI-restricted pARNS3 was inserted into the pACYM1 transfer vector.…”

Molecular Cloning and Expression of Bovine Viral Diarrhea Virus Nonstructural Protein 3 in Escherichia coli

“…Although, the extracts of virus infected cell cultures were formerly used in diagnostic ELISA kits as the detector antigens (28,29), several ELISAs have recently been developed to detect BVDV infections using recombinant NS3 protein (7,(16)(17)(18). Eukaryotic or prokaryotic recombinant NS3 may be used for this propose and this protein has shown high sensitivity and specificity for detection of BVDV infection in comparison with whole virus antigen (16,30).…”

“…Hence, production of a recombinant form of NS3 in large amounts is economically very essential and useful for constructing BVDV antibody ELISA kits. Although it has been shown that eukaryotic expression of NS3 increases the sensitivity and specificity of ELISA kits (18), prokaryotic expression of NS3 is still considered because it is simpler and less expensive than eukaryotic expression system.…”

“…Recombinant NS3 has been expressed and evaluated in different researches but a major problem of those expressions was that generally, this protein is insoluble and aggregates as inclusion bodies in recombinant bacterial cells. Thus, the expressed recombinant NS3 must be treated with urea to modify it a soluble protein for the convenient and effective protein purification (15)(16)(17)(18). Here, we used a different prokaryotic plasmid vector, pMAL-c2X, to express a soluble form of the protein with a high level of expression.…”