甜菜夜蛾拓扑异构酶I氨基酸突变对其DNA解旋活性的影响

【目的】为了探究甜菜夜蛾 Spodoptera exigua 拓扑异构酶I（topoisomerase I, Top I）氨基酸突变对其DNA解旋活性的影响。【方法】通过克隆甜菜夜蛾 Top I 基因，构建原核表达载体，采用完全重叠PCR定点突变技术，向甜菜夜蛾Top I 的V420, L530, A653和S729（根据人Top I 氨基酸序列编号）4个位点引入突变，将改造成功的重组 Top I 基因转化至大肠杆菌BL21 (DE3)中，诱导重组蛋白表达、纯化，测定Top I突变对其解旋活性的影响。【结果】完全重叠PCR能实现甜菜夜蛾 Top I 定点突变。重组蛋白在体外得到稳定的表达，表达产物经SDS-PAGE电泳分析在96.0 kDa处出现特异性条带。通过对重组蛋白分离纯化并测定对质粒pBR322解旋酶活性，发现引入V420I, L530P和A653T突变后Top I的比活力显著降低，而引入S729T突变后比活力与野生型蛋白无显著差异。【结论】本研究证明在甜菜夜蛾Top I中引入V420I, L530P和A653T突变后，其对底物pBR322的解旋活性显著降低，为后期探索甜菜夜蛾Top I的定点突变与其对喜树碱及其衍生物敏感性的关系奠定了基础。

Effects of amino acid substitutions of topoisomerase I on its DNA relaxation activity in Spodoptera exigua (Lepidoptera: Noctuidae)

【Aim】 This study aims to investigate the effects of amino acid substitutions of topoisomerase I (Top I) on its DNA relaxation activity in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae). 【Methods】 The NH₂ -terminally truncated Top I (Top70) from S. exigua was amplified and cloned into the BamH I-Sal I sites of the pGEX-4T-1. Mutant constructs substituted with V420I, L530P, A653T and S729T (numbered according to human Top I) were obtained by PCR-based site-directed mutagenesis using the wild construct pGEX-4T-1-Top70 as the template separately. The wild and mutated constructs were expressed in Escherichia coli strain BL21 (DE3). The GST fusion protein was purified using GSTrap 4B, and the DNA relaxation activity of the purified protein was detected with pBR322 as substrate. 【Results】 The wild and mutated pGEX-4T-1-Top70 expression vector was successfully constructed, and the cell line stably expressing Top70 was established. A 96.0 kDa specific protein band appeared when the expressed protein was analyzed with SDS-PAGE. Compared to the wild Top I, the mutated Top I with amino acid substitutions at positions V420I, L530P and A653T had significantly decreased DNA relaxation toward pBR322. However, the S729T amino acid substitution showed no significant impact on the catalytic efficiency of Top I. 【Conclusion】 These results suggest that V420T, L530P and A653T amino acid mutations in S. exigua remarkably decline the affinity of Top I to pBR322, providing important basic information for further investigating the sensitivity of Top I to the camptothecin and its analogous.