Improvement of recombinant baculovirus infection efficiency to express manganese superoxide dismutase in silkworm larvae through dual promoters of Pph and Pp10

Abstract

The silkworm, Bombyx mori, has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). There are several problems which will probably be the bottleneck for practical and industrial utilization of silkworm bioreactor. Traditionally, the recombinant virus should infect the larvae through individual dorsal injection by a syringe. This is a time- and labor-consuming procedure. This drawback has become a bottleneck for practical and industrial utilization of baculovirus expression system in the silkworm bioreactor. In this paper, we constructed a dual expression baculovirus to express the renovated polyhedron and target manganese superoxide dismutase (SOD) gene under P10 and polyhedron promoters, respectively, through oral infection. The results showed that the direct injection of recombinant rBacmid/BmNPV/SOD DNA with cellfectin reagent infected the silkworm larvae partially. When next batches of larvae were fed orally with hemolymph, which was collected from first batch of injected and infected larvae, the obvious symptom of infection was found and high target SOD was expressed. These results imply it is feasible to express target genes through combination of recombinant bacmid DNA injection and oral feeding by a dual expression bacmid baculovirus.

Designing Two Individual AcMNPV Polyhedrin-Plus Bac-to-Bac Expression System in order to Express GFP and CPV-VP2 in Insect Cells

“…The Bac-to-Bac requires less time to generate (2 weeks as opposed to 4-6 weeks), no need for plaque selection due to working based on blue/white selection, rapid and simultaneous isolation of several recombinant baculoviruses This method allows quick and simultaneous, and suitable for the expression of various proteins in structural and functional studies (23,(25)(26). Considering these advantages, here Bacto-Bac baculoviral system was set up to express VP2 of canine parvovirus in large-scale in insect cells.…”

“…The fl anking sites of the expression cassette (that contains the inserted gene) in the pFastBac vector are the right and left arms of Tn7 (Tn7R and Tn7L), and also this cassette is containing a resistance gene (gentamicin) and a SV40 poly A signal that forms a mini Tn7 transposon (23,(25)(26).…”

“…Bac-to-Bac baculoviral expression vector system (BEVS) is an effi cient and fast method to produce recombinant baculoviruses (23,(25)(26).…”

Human insulin gene expressing with Bombyx mori multiple nucleopolyhedrovirus (BmMNPV) expression system

“…Silkworms are large and easy to rear and manipulate and have a relatively short life cycle (approximately 7 days post-injection). Moreover, we used bacmid DNA developed in our laboratory for direct injection into silkworm larvae, thereby avoiding the preparation of high-titer viral stocks for infecting larvae (Yue et al 2008;Liu et al 2009).…”

Industrial Production of Superoxide Dismutase (SOD): A Mini Review

“…The yeast glyceraldehyde phosphate dehydrogenase promoter was used in the high level expression of human SOD in yeast [36]. The bacmid a baculovirus shuttle vector system used as a vector to overexpress Mn SOD in insects like Bombyx mori as a bioreactor [37]. The human liposarcoma (LSA) cells were used as a host for the synthesis of Mn SOD, but astoundingly it was secreted in the medium than being found in the mitochondria.…”