Production of Classical Swine Fever Virus Envelope Glycoprotein E2 as Recombinant Polyhedra in Baculovirus-Infected Silkworm Larvae

Abstract

Although, classical swine fever virus (CSFV) envelope glycoprotein E2 subunit vaccine has been developed using the baculovirus expression system, the expression of viral antigens in baculovirus-infected insect cells is often ineffective. Therefore, an alternative strategy to the traditional baculovirus expression system is needed that is more productive and effective. Here, we report a novel strategy for the large-scale production of a CSFV E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg/ml of hemolymph and 0.53 mg/larva at 6-days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedra elicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that this strategy can be used for the large-scale production of CSFV E2 antigen.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification

“…The results indicated that immunization with a chimeric protein induced a significant serum antibody reaction against Polyhedrin and the coat protein. This demonstrated that both were immunogenic and that the baculovirus protein perhaps increases the immune response as previously observed [17,22] for another baculovirus carrier protein and the Polyhedrin. Similar methods have already been developed and employed to purify target proteins [16-18]; however, the novelty presented here is the expression of a full-length protein in fusion with the Polyhedrin and the use of complete cadavers of caterpillars as bioreactors.…”

“…This feature avoided the polyhedral matrix formation and the nuclear localization by the chimeric protein. Previous research has used, besides the polh gene fused with a gene of interest, a second polh gene copy [14,17]. Although the presence of a second copy of polh could improve the chimeric crystal formation and nuclear localization, we observed that the presence of only one fused Polyhedrin-copy was sufficient to form a crystal structure.…”

Functional Virus-Like Particles Production Using Silkworm and Their Application in Life Science

“…Classical swine fever virus envelope glycoprotein, E2, fused with polyhedron from baculovirus has been expressed in silkworm larvae and E2 has been purified by solubilization of recombinant polyhedra. Virus-neutralizing activity is induced when purified E2 is used to immunize mice [77]. In this case, virus-neutralizing activity is induced after immunization with recombinant polyhedra.…”

Research Article An improved Bac-to-Bac/BmNPV technology expressing envelope E2 glycoprotein of classical swine fever virus (CSFV) in the silkworm, <i>Bombyx mori</i>

“…Meanwhile, recent findings have shown powerful immunogenicity among baculovirus expressing E2 glycoprotein (Dong and Chen 2007), thus, further proving for quick and economical production of vaccines in veterinary industry (Wu et al 2010). Later, Lee et al 2012 achieved production of recombinant E2 glycoprotein using BVES system in silkworm larvae (Lee et al 2012).…”