Expression of maedi-visna virus major core protein, p25: development of a sensitive p25 antigen detection assay.
The gene for the major core protein, p25, of maedi-visna virus (MVV) was cloned using a PCR (polymerase chain reaction) strategy employing primers designed for the insertion of the gene directly into yeast Ty-VLP expression vectors. In this system p25 is expressed as a fusion protein which self-assembles into virus-like particles (VLPs) due to interaction of the Ty A fusion partner. High levels (50-60 mg/l) of p25 fusion protein were produced, and p25 was recovered in soluble and highly pure form following cleavage from the Ty particle by Factor Xa protease digestion. The p25 protein produced in yeast is antigenically authentic, as defined by its reactivity with p25-specific antisera and its ability to elicit antibodies reactive with native viral p25 protein; although the cleaved, soluble form of p25 was found to be considerably more antigenic than the hybrid Ty-p25 VLP. Using this reagent anti-p25 monoclonal and polyclonal antibodies were generated. These sera and the p25 protein have been used to develop a sensitive MVV p25 detection assay. These reagents and assays will facilitate further studies of viral replication and immune response to the virus.
Reyburn
H T
HT
Department of Veterinary Pathology, University of Edinburgh, Summerhall, U.K.
Roy
D J
DJ
Blacklaws
B A
BA
Sargan
D R
DR
McConnell
I
I
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis*; D001483:Base Sequence; D002478:Cells, Cultured; D005814:Genes, Viral; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D015678:Viral Structural Proteins / Q000235:genetics; D014790:Visna-maedi virus / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D015678:Viral Structural Proteins