Abstract
A new baculovirus-silkworm multigene expression system named Bombyx mori MultiBac is developed and described here, by which multiple expression cassettes can be introduced into the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome efficiently. The system consists of three donor vectors (pCTdual, pRADM and pUCDMIG) and an invasive diaminopimelate (DAP) auxotrophic recipient E. coli containing BmNPV-Bacmid (BmBacmid) with a homologous recombination region, an attTn7 site and a loxp site. Two genes carried by pCTdual are firstly inserted into BmBacmid by homologous recombination, while the other eight genes in pRADM and pUCDMIG are introduced into BmBacmid through Tn7 transposition and cre-loxp recombination. Then the invasive and DAP auxotrophic E. coli carrying recombinant BmBacmid is directly injected into silkworm for expressing heterologous genes in larvae or pupae. Three structural genes of rotavirus and three fluorescent genes have been simultaneously expressed in silkworm larvae using our new system, resulting in the formation of virus-like particles (VLPs) of rotavirus and the color change of larvae. The VLPs were purified from hemolymph by ultracentrifugation using CsCl gradients, with a yield of 12.7 µg per larva. For the great capacity of foreign genes and the low cost of feeding silkworm, this high efficient BmMultiBac expression system provides a suitable platform to produce VLPs or protein complexes.
Highlights
Baculovirus expression system is one of the most ideal systems for routine production of recombinant eukaryotic proteins in insect cells, larvae and mammalian cells, which is widely-used in developing virus-like particles (VLPs) vaccine, displaying heterologous peptides or proteins, and transducing genes into mammalian cells [1,2]
We successfully develop a novel silkworm-based baculovirus multigene expression system, in which multiple proteins can be expressed simultaneously to produce protein complexes or VLPs efficiently
Construction of Bombyx mori (silkworm) nucleopolyhedrovirus (BmNPV)–silkworm multigene expression system Our BmNPV–silkworm multigene expression system consists of the modified recipient strain E. coli IBIDsw106MultiBmBac and three donor vectors including pCTdual, pRADM and pUCDMIG
Summary
Baculovirus expression system is one of the most ideal systems for routine production of recombinant eukaryotic proteins in insect cells, larvae and mammalian cells, which is widely-used in developing virus-like particles (VLPs) vaccine, displaying heterologous peptides or proteins, and transducing genes into mammalian cells [1,2]. Besides the traditional Autographa californica multicapsid nucleopolyhedrovirus-Spodoptera frugiperda 9 (AcMNPV-Sf9) cell line system, another highly efficient baculovirus expression system, named Bombyx mori (silkworm) nucleopolyhedrovirus (BmNPV)-silkworm larvae/pupae system, has been constructed to express heterologous genes. Both vp and DsRed genes were introduced into MultiBmBacmid through homologous recombination according to previous study [5]. The vp and eyfp were introduced from pUCDM-YFP-vp into BmBacmid (BmMutiBac-Red-VP2) by Tn7 transposition as described previously [6]. The vp and IERS-egfp were introduced from pUCDMIG-vp into BmBacmid (BmMutiBac-Red-VP2-YFP-VP6) through cre-loxp site specific recombination according to previous report [8]
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