Recombinant RuV E1 Mosaic

Stably transfected Chinese hamster ovary (CHO24S) cells were the source for Rubella virus-like particles (RVLP) containing all structural proteins (E1, E2, C and their dimers). RVLP are secreted from the CHO24S cells into the medium and the time-point for collecting the medium with the highest yield of > 100 kDa proteins (with 17 mg protein from 10 ml cell culture supernatant) was after 2 days of incubation. Different methods for RVLP isolation from the cell culture supernatants were assessed by SDS-PAGE and Western blotting (using sera positive or negative for Rubella virus (RV)-specific antibodies or an anti-E1 monoclonal antibody). A combination of membrane filtration with a rapid, novel gradient ultracentrifugation step (using Coomassie brilliant blue G crystals as adsorbens for RVLP that facilitated virus isolation) was the most suitable technique. 132 RV-positive human sera (RV IgG > 20 IU/ml by commercial ELISA) were tested by our "self made" immunoblot test stripes (using RVLP adsorbed to dye crystals as antigen) for the presence or absence of antibodies specific for RV structural proteins. 57.6% of these sera had antibodies against E1, E2 and C, 31% against E1 and C, and 1.5% against E1 only, whereas 3.8% had no RV specific antibodies and only 6.0% were equivocal which demonstrated that these "self made" test stripes can reliably differentiate RV antibody specificities.

The capsid protein of rubella virus was produced in baculovirus-infected Spodoptera frugiperda insect cells, with a polyhistidine affinity tag at the carboxy terminus. The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal-ion affinity chromatography, Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. The final yield was 5 mg of purified protein per liter of cell culture. Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen-antibody interaction study. A specific interaction between the two proteins was shown, Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies, (C) 1998 Academic Press.