Confirmation of the incorporation of BS protein in BVLPs.

<p>(A) Budding of BVLPs from insect cells. Sf21 cells were co-infected with recombinant baculovirus vAcBS and vAcME at a moi of 5. At 72 h post-infection, cells were harvested and fixed with 2% glutaraldehyde and then with 1% osmium tetroxide. Thin-section samples were prepared and stained with 1% uranyl acetate followed by examination under a transmission electron microscope. Arrows indicates BVLPs. Bar = 250 nm. (B) Western blot analysis of BVLPs. 5 µg purified BVLPs were analyzed by western blot using rabbit-derived anti-SARS-CoV antibody. An equivalent amount of purified human SARS CoV VLPs was run in parallel. Preimmune rabbit antiserum was used as negative control. Left part, band corresponds to S protein. Lane 1, negative control; lane 2, human SARS CoV VLPs; lane 3, BVLPs. Right part, band corresponds to E or M protein. Lane 1, human SARS CoV VLPs; lane 2, negative control; lane 3, BVLPs. The protein marker was Fermentas #SM0671. (C) Detection of BVLPs by immunogold labeling. The collodion-coated EM grids were loaded with purified BVLPs (left), SARS CoV VLPs (middle) or VLPs without BS or S protein (right) for 5 min. After removal of the excess sample solution, grids were incubated with antibody specific against BS2 protein for 1 h and then incubated with 15 nm gold conjugated anti-rabbit IgG.. The samples were stained with 2% PTA for 1 min, drained and examined under the EM. Arrow indicates the gold particles and triangle indicates the VLPs. Bar = 100 nm.