家蝇乙酰胆碱酯酶基因密码子优化及酶学特性表征

Codon Optimization and Characterization of Acetylcholinesterase Gene from Musca domestica

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区段GC含量至45%~55%。将优化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。以改进的Ellman法测定重组家蝇乙酰胆碱酯酶(mdAchE)酶学性质,通过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在毕赤酵母中实现高效表达,酶活力约8.82 U/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,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1.83×10-15μg/mL,远远低于其它诸如气相色谱法、生物传感器等方法测得的最低检测限(0.01μg/mL,4.14×10-6μg/mL)。本试验结果表明,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大的应用潜力。

The gene sequence of 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 gene was ligated into the expression vector pPIC9 K 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 md Ach E was analyzed by organophosphorus and carbamate pesticide inhibition assays. The mdAchE codon was optimized to achieve high expression in Pichia pastoris with an enzyme activity of approximately 8.82 U/m L. 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 1.83×10-15μg/m L, which was far lower than the lowest detection limit(0.01 μg/m L, 4.14×10-6μg/m L) measured by other methods such as gas chromatography, biosensors. The recombinant enzyme md Ach E has great application prospects in the detection of organophosphorus and carbamate pesticide residues.