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乙酰胆碱酯酶(ACh E)在神经信号传导过程中起重要作用,是氨基甲酸酯类和有机磷类农药的主要作用靶标.对朱砂叶螨ACh E进行体外表达和纯化,并分析其活性.将朱砂叶螨ace基因序列插入到原核高效表达载体p ET-30a中,构建表达载体p ET-30a/ace,转入表达菌株Escherichia coli BL21(DE3)中,IPTG诱导高效表达;镍柱纯化目的蛋白,经SDS-PAGE和Western Blot鉴定目的蛋白;以纯化的螨ACh E为抗原制备抗螨ACh E特异性抗体;改良的Ellman法测定螨ACh E活性.结果获得了相对分子质量(Mr)约为68×103的较高纯度的螨ACh E蛋白,检测大部分为可溶性表达,酶活检测具有乙酰胆碱酯酶活性,并制备了107效价的抗螨ACh E特异性抗体.采用重组ACh E(IC50=5.4 mmol/L)检测毒扁豆碱抑制活性比螨粗酶液(IC50=58.7 mmol/L)灵敏度提高11倍.本研究构建了螨ACh E体外高效表达载体并获得了具有较好活性的重组螨ACh E,可为抗ACh E杀螨剂的体外高通量筛选和以ACh E为靶标的农药残留检测奠定基础.
Acetylcholinesterase (AChE) plays an important role in the process of neural signal transduction and is the main target of carbamates and organophosphorus pesticides. ACh E is expressed and purified in vitro and its activity is analyzed. The ace gene sequence of Tetranychus cinnabarinus was inserted into the prokaryotic expression vector p ET-30a to construct the expression vector p ET-30a / ace which was transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The target protein , And the target protein was identified by SDS-PAGE and Western Blot.Anti-mite ACh E-specific antibody was purified with purified ACh E as antigen and ACh E activity was determined by modified Ellman method.Results The relative molecular mass (Mr) 68 × 103 higher purity mite ACh E protein, most of which were tested for soluble expression, acetylcholinesterase activity was detected by enzymatic activity, and anti-mite ACh E specific antibody was prepared with 107 titers.Using recombinant ACh E (IC50 = 5.4 mmol / L) showed that the inhibitory activity of physostigmine was 11 times higher than that of mite crude enzyme solution (IC50 = 58.7 mmol / L) .In this study, a highly efficient expression vector for mite ACh E was constructed and a recombinant with good activity Mite ACh E, anti-ACh E acaricide In vitro high-throughput screening and detection of pesticide residue with ACh E as the target.