Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system

Abstract

Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly established Bac-to-Bac/BmNPV Baculovirus expression system was used to overexpress the recombinant Mn-SOD enzyme in silkworm larvae. The hemolymph was collected from the infected larvae 96 h post-infection and subjected to a 12 % SDS-PAGE and Western blotting. A 18.0-kDa protein was visualized after rBacmid/BmNPV/SOD infection. The SOD enzyme activity was determined with a tetrazolium salt for detection of superoxide radicals generated by xanthine and xanthine oxidase and its peak appeared in 96 h post-infection with 2.7 times of the control larvae. The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for potential application in medicine.

Anti‐oxidation and immune responses in mice upon exposure to manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L

“…The fifth instar silkworm larvae expressed superoxide dismutase (SOD) 96 h post-infection with recombinant virus (rBacmid/BmNPV/SOD) were collected (Yue et al, 2006), and dried with a vacuum dryer (Brocher CHRIST Beta-16, Germany) under low temperature of À56 C. The dried larvae were homogenized to powder and stored at À20 C until use.…”

“…We used the silkworm, Bombyx mori larvae as a bio-reactor, and expressed the manganese superoxide dismutase (Mn-SOD) by the recombinant bacmid baculoviruses expression system (Yue et al, 2006). To study the function of superoxide dismutase and potential practical development, we investigated the antioxidative and immunological function of SOD in the plasma and liver organs of treated mice with SODincluding silkworm larvae powder, suggesting the silkworm larvae powder containing SOD play a positive role in antioxidation and immunity promotion.…”

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

“…The baculovirus expression system has been proven to be very efficient for heterologous protein production than that obtained in naturally producing mammalian cells. It can also be used to perform post-translational modifications identical to mammalian ones, we have used the BmMNPV expression system with silkworm larvae as hosts to express foreign proteins (Yue et al 2006;Miao et al 2006a, b). Silkworms are large and easy to rear and manipulate and have a relatively short life cycle (approximately 7 days post-injection).…”

Cloning and differential expression of manganese superoxide dismutase (Mn-SOD) of Trichinella pseudospiralis

“…However, only a few Mn-SOD have been cloned. They include those of bacteria (Vibrio mimicus, Escherichia coli, Bordetella bronchiseptica, Halobacterium halobium), nematodes (Onchocerca volvulus, Caenorhabditis elegans), trematode (Clonorchis sinensis), silkworm (Bombyx mori), and Chinese shrimp (Fenneropenaeus chinensis) (Henkle-Duhrsen et al 1995;Hunter et al 1997;Li et al 2005;Yue et al 2006;Zhang et al 2007). However, SOD genes from adenophorean nematodes have not been studied extensively.…”