朱砂叶螨乙酰胆碱酯酶的表达纯化及其酶活测定条件优化

Expression and purification of Tetranychus cinnabarinus acetylcholinesterase and its enzyme assay optimization

乙酰胆碱酯酶(Acetylcholinesterase,AChE)是生物体内保证神经信号正常传递的关键酶,是有机磷和氨基甲酸酯类农药的作用靶标。现研究拟采用发酵罐培养含pET-30a/ace的表达菌株E.coli BL21(DE3),大量表达纯化螨AChE,利用单因素试验和正交试验优化AChE活性的测定条件。结果表明:发酵培养的重组菌株大量表达约为68kD的朱砂叶螨AChE蛋白。底物浓度、温度、pH和酶浓度等各测定因素对朱砂叶螨乙酰胆碱酯酶活性存在显著影响。确定朱砂叶螨乙酰胆碱酯酶活性测定的最佳反应条件为:底物浓度1mmol/L,pH值7,温度35℃,酶浓度0.5mg/mL。通过体外发酵培养纯化获得了大量的朱砂叶螨AChE重组蛋白,确定了螨AChE活性测定的最佳条件,为进一步研究其酶学性质以及AChE抑制剂的体外筛选奠定基础。

Acetylcholinesterase(AChE)plays a key role in normal nerve impulse transmission,and is the main target of organophosphate and carbamate insecticides.The aim of this study is to produce recombinant AChE of Tetranychus cinnabarinus by fermentation,and optimize the assay conditions in measuring AChE activity by single-factor test and orthogonal experiment.The results showed that high quality of the recombinant AChE protein was obtained with a molecular weight of 68 kD.Several factors including temperature,pH,enzyme concentration and substrate concentration,affect the reaction rate of AChE reaction.The optimal conditions of AChE reaction are as follows:substrate concentration 1mmol/L,pH 7,reaction temperature35℃,and enzyme concentration 0.5mg/mL.The obtained T.cinnabarinus AChE proteins with its reaction conditions optimized lay a solid foundation for screening acetylcholinesterase inhibitors.