A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus

Abstract

The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms.

Gene delivery and gene expression in vertebrate using baculovirus Bombyx mori nucleopolyhedrovirus vector

“…Human serum was obtained from Sigma-Aldrich (Deisenhofen, Germany). The reBmBac vector was constructed and prepared in our lab [ 42 ]. Expression of EGFP was analyzed by western blotting using the rabbit polyclonal anti-EGFP antibody (ab6556, Abcam, UK).…”

“…The recombinant Bm NPVs for reporter gene delivery, reBm-luc and reBm-EGFP, were prepared by co-transfection in Bm 5 cells with reBmBac and pVLCMV-luc or pVLCMV-EGFP as described [ 42 , 43 ], and purified by plaque screening [ 44 ]. Fifth instar silkworm larvae were used to amplify the viruses; they were injected with recombinant Bm NPVs (approximately 10 5 plaque forming units, p.f.u.).…”

IFN-γ establishes interferon-stimulated gene-mediated antiviral state against Newcastle disease virus in chicken fibroblasts

“…We have previously demonstrated the successful construction and development of a recombinant B. mori Bacmid (reBmBac) expression system [41]. The silkworm reBmBac gene expression system was used to construct and express recombinant chIFN-γ (GenBank: NM_205149.1; Gallus gallus).…”

“…Blank silkworm blood treated in the same way was loaded as the negative control. After electrophoresis and transfer onto PVDF membrane, recombinant chIFN-γ expression was analyzed by western blot analysis using anti-IFN-γ antibody (Abcam, Cambridge, USA), according to the Protein Blotting Guide (Bio-Rad, Hercules, USA), as previously described [41].…”

“…BEVS was used for the construction and expression of chIFN-γ. A Luciferase reporter gene was used to assess the expression efficiency as previously described [41]. Co-transfection of the pVL1393-luc plasmid and reBmBac DNA in Bm5 cells are presented in Fig.…”