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

Abstract

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.

Construction of a Baculovirus-Silkworm Multigene Expression System and Its Application on Producing Virus-Like Particles

“…However, the first two genes in pCTdual must be integrated into MultiBmBacmid using homologous recombination to remove the Gm R , whereas the other eight genes can be introduced into BmBacmid through cre-loxp and mini-Tn7 transposon methods simultaneously. The efficiency of transferring genes from pCTdual to BmBacmid is 99.8%, and it is sufficient to ensure the positive recombinant identification with almost 100% success when combining the Gm sensitive testing [6]. The background of transposition using pRADM and attTn7-blocked host is negligible, and the white colonies are sure to be positive at 100% efficacy when the blue-white screening is still preserved [7].…”

“…Plasmids pML291, pcp15 and pcp20 were gifts from Dr. Li [17], and spectinomycin ( spe )-resistance plasmid pGB2Ωinv–hly (containing both hly and inv genes) was provided by Prof. Courvalin [18]. Plasmids pBlock, pRCDM and pCTdual, as well as the modified BmBacmid with gentamycin resistance gene between two I-sce I sites were constructed at our previous study [6], [7]. Plasmids pUCDM and pFBDM were from Prof. Richmond [8].…”

“…BmBacmid-Gm, constructed previously by introducing a Gm R cassette flanking two I-Sce I sites into the p10 and p74 locus of the original BmNPV-Bacmid through homologous recombination, was transformed into E. coli SW106 [6]. After the induction of lambda red-gam recombinase at 42°C for 15 minutes, the electro-competent cells of SW106 containing BmBacmid-Gm were prepared and stored as previous description [16].…”

“…Until today, great efforts have been made for efficiently constructing recombinant BmNPV, including the BmNPV-based Bac-to-Bac system [4], [5], the mating-assisted genetically integrated cloning (MAGIC) method [6] and a method based on zero-background Tn7-mediated transposition in E. coli [7]. Other improvements relating to the baculovirus expression system also have been presented, such as utilizing cysteine protease and chitinase-deficient Bacmid to improve recombinant protein production and keep its stability [8], [9], as well as a transfectant-free method by directly infecting insect cells or injecting silkworm larva with invasive E. coli containing recombinant Bacmid [10], [11].…”

“…Another interesting multigene expression method in AcNMPV was performed using repeated homologous recombination and cre-loxp recombination to express up to 8 foreign proteins from 8 loci [12]. As some improvements on the construction of recombinant BmNPV have been made by us [6], [7], [10], [11], we are going to combine these different methods to establish another efficient MultiBac system based on BmNPV and silkworm. We have ever expressed rotavirus-like particles in cultured BmN cells by using the original vectors derived from MultiBac and the traditional method of recombinant baculovirus construction [13].…”