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 cl...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2006-11, Vol.73 (1), p.181-186
Main Authors: Yue, Wanfu, Miao, Yungen, Li, Xinghua, Wu, Xiaofeng, Zhao, Aichun, Nakagaki, Masao
Format: Article
Language:English
Subjects:
Animals
Bac-to-bac/BmNPV baculovirus expression system
Baculoviridae - genetics
Baculovirus
Biological and medical sciences
Biotechnology
Blotting, Western
Bombyx - enzymology
Bombyx - genetics
Bombyx mori
Cloning
Cloning, Molecular
DNA, Complementary
Electrophoresis, Polyacrylamide Gel
Enzymatic activity
Enzymes
Fundamental and applied biological sciences. Psychology
Genetic Vectors
Hemolymph - enzymology
Hydrogen peroxide
Infections
Larva - enzymology
Larvae
Manganese
Manganese superoxide dismutase (Mn-SOD)
Molecular Weight
Oxidation
Reverse Transcriptase Polymerase Chain Reaction
RNA - genetics
RNA - isolation & purification
Sequence Analysis, DNA
Silkworm larvae (Bombyx mori L.)
Superoxide Dismutase - biosynthesis
Superoxide Dismutase - genetics
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Summary: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.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-006-0462-y