466 et al. 2007) and pancreatic necrosis virus (Shivappa et al. 2005). Recently, Li et al. (2008) reported that the capsid proteins of foot-and-mouth disease virus (FMDV) were produced in the hemolymph of silkworm larvae, and cattle vaccinated with hemolymph, diluted 30-fold, were pro- tected from challenge with FMDV. The ability of BmNPV to display gp64 fusion proteins also offers the possibility of using this silkworm as a host for the production and display of a wide range of antigenically important proteins on the cell or virus surface. Using silkworm larvae, rather than insect cell lines, as a host for recombinant virus amplification and recombinant protein production has advantages, in terms of their size and easiness to rear. The purified virus particles displaying the cloned foreign protein on the viral envelope could be used directly for functional analysis, without isolating the proteins. Since the presence of the gp64 fusion protein did not alter the growth and yield of the virus, the availability of the gp64-fusion-protein-displayed virus will also provide insight into the virus–host cell interaction, and this system may provide a fundamental platform for large-scale vaccine production. The BmNPV- based system is an economic process for the large-scale production of surface-displayed baculoviruses and safe subunit vaccines for animals, since the silkworm larvae, which are easy to rear on a synthetic or natural diet, can be used instead of the cultured cell lines. Appl Microbiol Biotechnol (2010) 85:459–470 Expression of pharmaceutically relevant proteins, including cell/viral surface proteins The infection of silkworms with recombinant BmNPV virus is a very attractive strategy for protein production, especially for cell surface receptors and secreted proteins, which often require posttranslational modifications, includ- ing sugar modifications. Silkworm expression is used for the expression of pharmaceutically relevant proteins. Immune cell and viral surface molecules and guanine nucleotide-binding protein (G protein) coupled receptors (GPCR) are the main drug targets because they regulate a broad range of physiological functions (Kristiansen 2004). The production of cell surface proteins using the silkworm- BmNPV expression system is summarized in Table 2. The expression levels ranged from several micrograms to 0.5 mg per larva. The proteins normally had signal sequences and thus were secreted in the hemolymph when properly processed. The obtained proteins were basically functional, as confirmed by either ligand binding assays or ELISA. Human killer cell immunoglobulin-like receptor, KIR2DL1, which regulates natural killer cell function by recognizing human leukocyte antigen C molecules, was expressed successfully (Sasaki et al. 2009). The expression level was high, 0.2 mg/larva, either by direct injection of bacmid DNA or by injection of collected body fluid containing the recombinant virus. Furthermore, the protein was easily purified using Ni-NTA affinity chromatography. Expression level Table 2 Expression of cell/viral surface proteins and GPCRs in silkworm Receptor Gene transfer Yield Application Functional assay Function Reference KIR2DL1 IL4-Rα IL4-Rα-Fca IL13-Rα1 IL13-Rα1-Fca Influenza hemagglutinin Nociceptin receptor (Giα1)b µ-Opioid receptors BmNPV Bacmid ~0.2 mg/ larva BmNPV 0.11 mg/ml hemolymph 0.053 mg/ml hemolymph 0.55 mg/ml hemolymph 0.33 mg/ml hemolymph BmNPV 0.4–4 μg/ larva BmNPV bacmid Transgenic Expression of immune cell and viral surface receptors using silkworm SPR surface plasmon resonance analysis a Fc fusion proteins b Gα-fusion proteins SPR, sugar characterization SPR, sugar characterization Gel filtration ELISA Not determined 150–250 ng/ larva [35S]GTPγS binding assay (EC50=9.3 ± 3.4 nM) [3H]diprenorphine saturation analysis (Kd=1.4~2.1 nM) Confirmed Confirmed Sasaki et al. 2009 Honjo et al. 2008 Not confirmed Sugiura et al. 2001 Kajikawa et al. 2009 Tateno et al. 2009