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Expression of an omega class glutathione S-transferase gene from Bombyx mandarina in the Sf9 cell
Author(s): 
Pages: 996-1001
Year: Issue:  10
Journal: ACTA ENTOMOLOGICA SINICA

Keyword:  野桑蚕谷胱甘肽S-转移酶基因克隆表达杆状病毒表达系统Sf9细胞;
Abstract: 谷胱甘肽S-转移酶(glutathione S-transferases,GSTs)是昆虫的重要解毒酶之一.为了研究野桑蚕Bombyx mandarina中谷胱甘肽S-转移酶在真核表达系统中的表达情况.本研究通过RT-PCR从野桑蚕中肠中获得GST-Omega1基因的cDNA序列,该基因的开放读码框为771 bp,编码256个氨基酸.对推导的氨基酸序列用NCBI的蛋白质Conserved Domains工具进行在线分析,结果显示GST-Omega1的氨基酸序列中具有Cys38和8个GSH结合位点的Omega类基因保守序列.对所获得的基因克隆进表达载体pFastBacHT b中获得pFast-GST-Omega1,将其转化DH10Bac感受态细胞,获得Bac-GST-Omega1重组病毒DNA,用脂质体法转染草地贪夜蛾Sf9细胞,获得重组病毒.对表达产物经SDS-PAGE和Western blotting分析,能检测到一条分子量约为33 kD的特异性条带,与推导的融合蛋白大小相符,该目的蛋白的表达量占总蛋白的14.4%.目的蛋白经His·Bind树脂纯化,用Lineweaver-Burk作图法测定其Km和Vmax,结果显示其Km为2.81 μmol/L,Vmax为2.70μmol/(mg·min).
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