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朱砂叶螨乙酰胆碱酯酶的原核表达及其活性分析 被引量:2

In vitro expression and activity analysis of acetylcholinesterase (ACHE) of Tetranychus cinnabarinus
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摘要 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.图8表1参19 Acetylcholinesterase (ACHE) plays an important role in the process of neural conduction, and is the major targetof organophosphate and carbamate insecticides. In order to characterize Tetranychus cinnabarinus ACHE, we studied the in vitro expression and purification of AChE of T. cinnabarinus, and determined its activity and sensitivity to physostigmine. The recombinant plasmid pET-30a/ace was constructed by subcloning the AChE gene into pET-30a. The expression of AChE in E. coli BL21 (DE3) was detected by SDS-PAGE and Western Blotting. Then the AChE recombinant protein was purified by Ni-NTA column. The activity of AChE was assayed by the improved Ellman method. The pET expression vector of T. cinnabarinus AChE was constructed, and high quality of the mite recombinant AChE protein was obtained with a molecular weight (M0 of 68 x 10^3 detected by SDS-PAGE and Western Blotting. The recombinant AChE protein had AChE activity, and the specific AChE antibody was obtained. Furthermore, physostigmine was found to be much more sensitive to recombinant AChE protein (IC50 = 5.4 mmol/L) than mite crude extracts (IC50 = 58.7 mmol/L). The high quality of AChE recombinant protein obtained in this study can be used for screening acetylcholinesterase inhibitors and detecting anti-AChE insecticide residues.
作者 彭博 卜春亚 师光禄 李金玲 冯晓姣 韩晶玉 高品 王有年 PENG Bo, BU Chunya , SHI Guanglu, LI Jinling, FENG Xiaojiao, HAN Jingyu, GAO Pin WANG Younian( Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China 2College of Biological Science and Engineering, Beijing University of Agriculture, Beijing 102206, China 3 Kep, Laboratory of Urban Agriculture (North) of Ministry of Agriculture . R. China, Beijing University of Agriculture, Beijing 102206, China)
出处 《应用与环境生物学报》 CAS CSCD 北大核心 2015年第2期215-220,共6页 Chinese Journal of Applied and Environmental Biology
基金 国家自然科学基金项目(31200483)、北京市自然科学基金项目(6122005)、教育部科学技术重点研究项目(212001)、北京市教育委员会科技计划面上项目(KM201210020001)和北京市教育委员会平台项目(KCT2014001)资助
关键词 朱砂叶螨 乙酰胆碱酯酶 原核表达 纯化 半数抑制浓度 Tetranychus cinnabarinus acetylcholinesterase (ACHE) prokaryotic expression purification half maximalinhibitory concentration (IC50)
作者简介 通讯作者Correspondingauthor(E-mail:buchunya@163.com)
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同被引文献35

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