A highly efficient method for the generation of a recombinant Bombyx mori nuclear-polyhedrosis-virus Bacmid and large-scale expression of foreign proteins in silkworm (B. mori) larvae.

In the post-genomic era, one of the challenges and a source of competition is the development of high-throughput, large-scale and low-cost eukaryotic cDNA cloning and expression systems. The baculovirus expression system is the most popular one and plays an important role in the high-level expression of eukaryotic proteins. In the present study, a convenient, rapid and highly efficient method for the construction of recombinant BmNPV (Bombyx mori nuclear polyhedrosis virus)-Bacmid vector (BmBacmid) for low-cost protein expression in silkworm (B. mori) larvae was established by using the MAGIC (mating-assisted genetically integrated cloning) strategy. By simply mixing the donor bacteria strain containing the constructed donor vector pCTdual harbouring foreign genes and the recipient strain containing modified BmBacmid, 99.8% positive recombinant BmNPV-Bacmids were obtained. Reporter genes egfp (enhanced green fluorescent protein gene) and DsRed (Discosoma sp. red fluorescent protein gene) and target gene man (beta-mannanase gene) encoding beta-mannanase were expressed in the silkworm larvae of B. mori at high level by injection of recombinant BmBacmid DNA directly with the standard calcium phosphate transfection procedure. The possibility of constructing a high-quality baculovirus cDNA library by transferring an ordinal plasmid cDNA library into the recipient BmBacmid in Escherichia coli was explored.