Production of scFv-displaying BmNPV in silkworm larvae and its efficient purification.

Baculovirus-display technology utilizing the gp64 envelope protein has been developed. A simple and efficient process to separate the virus from the majority of the protein contaminants may be needed for the future demand of pure and functional baculovirus vectors ideal for vaccine- and gene-delivery applications. In the present study, using Bombyx mori (silkworm) larvae as a host, scFv (single-chain variable fragment)-surface displaying recombinant baculovirus production and its purification from silkworm larval haemolymph by SEC (size-exclusion chromatography) were demonstrated. The amounts of scFv were 4-8 μg/ml in the haemolymph. The scFv-gp64 fusion protein was confirmed to be incorporated into the cell membrane and the BmNPV (B. mori nucleopolyhedrovirus) surface by immunofluorescence microscopy and Western blotting. rBmNPV (recombinant BmNPV) was purified to higher purity by SEC using Sephacryl S-1000 column chromatography than by sucrose-density-gradient centrifugation. The recovery of purified rBmNPV was 22.2%, and the virus purity in the SEC fraction was increased 269-fold compared with its purity in haemolymph. Judging from the results of ELISA, approx. 0.9% of the total baculovirus-particle proteins were occupied by scFv on their surface. A BmNPV-based silkworm-larval system is suitable for large-scale production of baculovirus-surface-displayed proteins or peptides in comparison with a cell-culture system. The present study will be useful for future BmNPV-application studies for gene delivery and vaccine trials.