Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system
Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system
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DOI：
10.1007/s00253-006-0462-y
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发表时间：
2006-11-01
影响因子：
5
通讯作者：
Nakagaki, Masao
中科院分区：
文献类型：
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作者：
Yue, Wanfu;Miao, Yungen;Nakagaki, Masao
超氧化物歧化酶（SOD）是催化超氧阴离子歧化为分子氧和过氧化氢的金属酶，因此形成细胞抗氧化防御机制的关键部分。本文以家蚕脂肪体总RNA为材料，通过RT-PCR方法克隆648 bp的Mn-SOD cDNA片段并测序。此外，利用新建立的Bac-to-Bac/BmNPV杆状病毒表达系统在家蚕幼虫中高效表达重组MnSOD酶。在感染后96小时从感染的幼虫收集血淋巴，并进行12% SDS-PAGE和Western印迹。感染rBacmid/ BmNPV/SOD后，可观察到一个分子量为18.0 kDa的蛋白。以黄嘌呤和黄嘌呤氧化酶产生的超氧阴离子自由基为指标，用四氮唑盐法测定SOD酶活性，其最高峰出现在感染后96 h，是对照的2.7倍。家蚕提供的大量SOD的可用性将极大地促进这种蛋白质在医学上潜在应用的未来研究和测试。
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 MnSOD 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.