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

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.

Possible functional co-operation of palindromes hr3 and hr4 in the genome of Cydia pomonella granulovirus affects viral replication capacity

“…This technology has been extended to several baculoviruses, e.g. Helicoverpa armigera nucleopolyhedrovirus (Wang et al, 2003), Spodoptera exigua multiple nucleopolyhedrovirus (Pijlman et al, 2002) and Bombyx mori nucleopolyhedrovirus (Yao et al, 2012). Hilton et al (2008) constructed a bacmid of CpGV also using the unique Pac I site for inserting the transfer vector cassette that derived from the AcMNPV Bac-to-Bac system.…”

Construction and Characterization of Virus-Like Particles: A Review

“…Interestingly, baculovirus-based vectors can also be used with the whole insect larvae, including silkworm larvae [51], as a cheaper alternative to produce VLPs [60]. The most recent baculovirus-silkworm multigene expression system insures the simultaneous expression of six different transgenes from a single recombinant baculovirus [61].…”

The Mulberry Silkworm—A New Source of Bioactive Proteins

“…It is also a good model organism for pest control studies [59] and a bioreactor for recombinant eukaryotic protein production [60]. The long-term expression of a human recombinant proteins in the silk glands had been demonstrated following gene delivery with a recombinant baculovirus [61,62]. The protein production capacity of mulberry silkworms exceeds that of any other industrial system.…”