Premium
Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac‐to‐Bac/BmNPV baculovirus
Author(s) -
Miao Y.G.,
Zhao A.C.,
Zhang Y.S.,
Nakagaki K.,
Meng Y.,
Zhao T.F.,
Nakagaki M.
Publication year - 2006
Publication title -
journal of applied entomology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.795
H-Index - 60
eISSN - 1439-0418
pISSN - 0931-2048
DOI - 10.1111/j.1439-0418.2006.01051.x
Subject(s) - hemolymph , biology , bombyx mori , recombinant dna , baculoviridae , bombyx , bombycidae , microbiology and biotechnology , silk , gene , instar , larva , virology , botany , genetics , spodoptera , computer science , operating system
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.