文章摘要
基于密码子优化的家蝇乙酰胆碱酯酶在毕赤酵母中的表达及其酶学性质分析
Expression and characterization of Musca domestica L. acetylcholinesterase based on codon optimization in Pichia pastoris
投稿时间:2019-01-13  修订日期:2019-01-13
DOI:
中文关键词: 乙酰胆碱酯酶  密码子优化  异源表达  有机磷农药  氨基甲酸酯类农药
英文关键词: acetylcholinesterase, codon optimization, heterologous expression, organophosphorus pesticides, carbamate  pesticides
基金项目:乙酰胆碱酯酶基因的挖掘及分子改造
作者单位E-mail
袁巧敏 河北农业大学食品科技学院 772477575@qq.com 
卢海强 河北农业大学食品科技学院  
黄蕾 河北农业大学食品科技学院  
谷新晰 河北农业大学食品科技学院  
李 晨 河北农业大学食品科技学院  
田洪涛 河北农业大学食品科技学院 tht631022@163.com 
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中文摘要:
      基于密码子通用性及不同物种密码子使用频率差异原则,对家蝇乙酰胆碱酯酶的基因序列进行优化,旨在提高其在毕赤酵母(Pichia pastoris)中的表达量,增强其应用前景。将基因序列中的低频密码子更换为毕赤酵母偏好的密码子,同时调整乙酰胆碱酯酶各个区段GC含量至45%-55%。将优化后的家蝇乙酰胆碱酯酶连接至表达载体pPIC9K并转入毕赤酵母GS115中,摇瓶培养。以改进后的Ellman 法测定重组家蝇乙酰胆碱酯酶(Musca domestica L. acetylcholinesterase, mdAchE)酶学性质,有机磷及氨基甲酸酯类农药抑制试验分析重组酶mdAchE的生物活性。家蝇乙酰胆碱酯酶密码子优化后实现了在毕赤酵母中的高效表达,酶活力约为8.82U/mL。抑制试验显示,重组酶mdAchE能够被马拉硫磷等7种有机磷农药和克百威等8种氨基甲酸酯类农药抑制,最低抑制浓度IC15值为1.83E-15μg/mL,远远低于其他诸如气相色谱法、生物传感器等方法测得的最低检测限(0.01μg/mL、4.14×10-6μg/mL)。上述实验结果表明重组乙酰胆碱酯酶mdAchE在有机磷及氨基甲酸酯类农药残留的检测领域具有较强的应用潜力。
英文摘要:
      the gene sequence of Musca domesticaSL. acetylcholinesterase was optimized based on codon versatility and the principle of difference codon usage frequency of different species, aiming to increase its expression in Pichia pastoris and enhance its application prospects. The low frequency codons in the gene sequence were replaced with Pichia pastoris preferred codons, while the GC content of each segment of the acetylcholinesterase was adjusted to 45%-55%. The optimized Musca domestica acetylcholinesterase was ligated into the expression vector pPIC9K and transferred into Pichia pastoris GS115, and cultured in shake flasks. The enzymatic properties of the recombinant enzyme mdAchE were determined by the modified Ellman method, and the bioactivity of the recombinant enzyme mdAchE was analyzed by organophosphorus and carbamate pesticide inhibition assays. The mdAcE codon was optimized to achieve high expression in Pichia pastoris with an enzyme activity of approximately 8.82 U/mL. The inhibition test showed that the acetylcholinesterase could be inhibited by seven organophosphorus pesticides such as malathion and eight carbamate pesticides such as carbofuran. The minimum inhibitory concentration IC15 value was 1.83E-15μg/mL, which was far lower than the lowest detection limit (0.01μg/mL, 4.14×10-6μg/mL) measured by other methods such as gas chromatography, biosensors. The recombinant enzyme mdAchE has great application prospects in the detection of organophosphorus and carbamate pesticide residues.
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