Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac‐to‐Bac/BmNPV baculovirus

Abstract

The silkworm has become an ideal multicellular eukaryotic model system for basic research. The major advantages of expressing foreign genes in silkworm larvae are the low cost of feeding, the extremely high levels of expression achievable compared with expression in cell lines and increased safety because the baculovirus is noninfectious to vertebrates. In this study, we used a recently developed Bombyx mori Nucleopolyhedrovirus (BmNPV) bacmid to express the spider flagelliform silk gene in silkworm larvae. The recombinant bacmid baculoviruses (rBacmid/BmNPV/Flag) were introduced into the first-day larvae of the fifth instar by subcutaneous injection. The worms presented symptoms typical of NPV infection from 72 h after injection compared with control. The haemolymph was collected from the infected larvae 120 h post-infection and the recombinant 6· His-tagged Flag protein was purified by the Ni-NTA spin kit under denaturing conditions with 8 m urea. A 37.0-kDa protein was visualized both in rBacmid/BmNPV/Flag-infected haemolymph and eluting fraction. The results showed that the Bac-to-Bac/BmNPV baculovirus expression system is an efficient tool to express the target gene in silkworm larvae, which takes only 7-10 days for generating recombinant baculovirus, compared with the traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses.

Expression of two heterologous proteins depends on the mode of expression: comparison of in vivo and in vitro methods

“…These include faster and higher production of protein and lower cost [5][6][7]. Kost et al [1] suggest that this system has not been widely exploited because of a lack of experience in rearing and maintaining larvae in laboratory conditions.…”

Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase

“…bioreactors) is required and there are several problems associated with the scaling up that negatively affects the specific production of proteins (Ikonomou et al 2003). Therefore, large-scale production using insect larvae (Lepidoptera: Noctuidae) as expression hosts is becoming a more attractive strategy as the manufacturing cost could be reduced up to four hundred times in comparison with cell cultures (Wu et al 2002;Seghal et al 2003;Maio et al 2006;Perez-Filgueira et al 2007).…”

High-level expression and purification of recombinant wheat germ agglutinin in Rachiplusia nu larvae

“…Despite these facts, BEVS versatility allows using insect larvae (Lepidoptera, Noctuidae family) for fast and low-cost production of a recombinant protein. The use of larvae as "biofactories" could reduce the manufacturing costs up to four hundred times in comparison with insect cell cultures [21][22][23][24]. On the other hand, the cost of the downstream processing step could increase significantly when using insect larvae as expression hosts, especially when several purification steps are required [25][26][27].…”