朱砂叶螨乙酰胆碱酯酶的原核表达与纯化 - 应用与环境生物学报.doc

朱砂叶螨乙酰胆碱酯酶的原核表达及其活性分析* 彭 博1,3 卜春亚2,3**师光禄2,3 李金玲1,3 冯晓姣1,3 韩晶玉1,3 高 品1,3 王有年2,3 1 北京 102206 2北京农学院生物科学与工程学院 北京 102206 3农业部都市农业(北方)重点开放实验室 北京农学院 北京 102206 摘 要 乙酰胆碱酯酶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作用靶标。朱砂叶螨乙酰胆碱酯酶本研究将朱砂叶螨ace序列插入到pET-30a中，构建了表达载体pET-30a/ace转入表达菌株E. coli BL21（DE3）中，IPTG诱导高效表达。纯化，SDS和Western Blot鉴定改良的Ellman法测定活性获得了分子量约为68 kD的蛋白。重组AChE毒扁豆碱比粗酶液为剂筛选和农药检测奠定了基础朱砂叶螨；乙酰胆碱酯酶原核表达；纯化 CLC S433.7 n vitro expression and activity analysis of acetylcholinesterase (AChE) of Tetranychus cinnabarinus * PENG Bo1,3, BU Chunya 2,3**, SHI Guanglu 2,3, LI Jinling1,3, FENG Xiaojiao1,3, HAN Jingyu1,3, GAO Pin1,3 WANG Younian 2,3 1Plant Science and Technology College, Beijing University of Agriculture,Beijing 102206, China 2College of Biological Science and Engineering, Beijing University of Agriculture,Beijing 102206, China 3Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture P. R. China, Beijing University of Agriculture, Beijing 102206, China Abstract Objectives: Acetylcholinesterase (AChE) plays an important role in the process of neural?conduction, and it is the major target of organophosphate and carbamate insecticides. In order to characterize Tetranychus cinnabarinus AChE and high-throughput screen AChE inhibitors in the near future, we conducted in vitro expression and purification of AChE of T. cinnabarinus, and determined its activity and its sensitivity to physostigmine. Methods: 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 SDSand Western Bloting. Then the AChE recombinant protein was purified by?Ni-NTA column. The activity of AChE was assayed by the improved Ellman method. Results: The pET expression vector of T. cinnabarinus AChE was constructed, and high quality of the mite recombinant AChE protein was got with a molecular weight of 68 kD detected by SDSand Western Blotting. The recombinant AChE protein has AChE activity,