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Preparation of divalent antigen-displaying enveloped virus-like particles using a single recombinant Bombyx mori
Tatsuya Kato1, Yuki Machida2, Kenshin Takemura3
1Laboratory of Biotechnology, Department of Agriculture, Graduate School of Integrated Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, Japan; Laboratory of Biotechnology, Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Suruga-ku, Shizuoka, Japan; Laboratory of Biotechnology, Green Chemistry Research Division, Research Institute of Green Science and Technology, Shizuoka University, Ohya 836, Suruga-ku, Shizuoka, Japan.
Abstract:
Silkworms have been used as a host for the production of recombinant proteins in a baculovirus expression system using Bombyx mori nucleopolyhedrovirus (BmNPV). To coexpress several recombinant proteins, a silkworm must be coinfected with several recombinant BmNPVs, which requires a difficult DNA manipulation procedure. In this study, we constructed recombinant BmNPVs containing three expression cassettes, Rous sarcoma virus (RSV) Gag protein, surface antigen 1 of Neospora caninum (NcSAG1) and SAG1-related sequence 2 of N. caninum (NcSRS2), by Gibson assembly and the Bac-to-Bac system, designated BmNPV/SAG-SRS-Gag and BmNPV/SAG-Gag-SRS. BmNPV/SAG-SRS-Gag was expressed in silkworms and characterized. NcSAG1 and NcSRS2 were purified with RSV Gag proteins using sucrose density gradient centrifugation and affinity chromatography. RSV Gag formed virus-like particles (RSV-LPs) at a diameter of 20-30 nm based on transmission electron microscopy (TEM). Immuno-TEM analysis showed that both NcSAG1 and NcSRS2 were displayed on the surface of the RSV-LPs. These results indicate that RSV-LPs displaying two different kinds of proteins were produced in the hemolymph of silkworm larvae by the single polycistronic strategy. This expression platform is efficient for generating multiantigen-displaying VLPs and facilitates the development of vaccines against infectious diseases.
