Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system.
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.
Yue
Wanfu
W
College of Animal Sciences, Zhejiang University, Hangzhou, 310029, People's Republic of China.
Miao
Yungen
Y
Li
Xinghua
X
Wu
Xiaofeng
X
Zhao
Aichun
A
Nakagaki
Masao
M
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006458:Hemolymph / Q000201:enzymology; D007814:Larva / Q000201:enzymology; D008970:Molecular Weight; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics
D018076:DNA, Complementary; D012313:RNA; D013482:Superoxide Dismutase