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[1]彭博,卜春亚,师光禄,等.朱砂叶螨乙酰胆碱酯酶的原核表达及其活性分析[J].应用与环境生物学报,2015,21(02):215-220.[doi:10.3724/SP.J.1145.2014.08008]
 PENG Bo,BU Chunya,SHI Guanglu,et al.In vitro expression and activity analysis of acetylcholinesterase (AChE) of Tetranychus cinnabarinus[J].Chinese Journal of Applied & Environmental Biology,2015,21(02):215-220.[doi:10.3724/SP.J.1145.2014.08008]
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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()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
21卷
期数:
2015年02期
页码:
215-220
栏目:
研究论文
出版日期:
2015-04-25

文章信息/Info

Title:
In vitro expression and activity analysis of acetylcholinesterase (AChE) of Tetranychus cinnabarinus
作者:
彭博 卜春亚 师光禄 李金玲 冯晓姣 韩晶玉 高品 王有年
1北京农学院植物科学技术学院 北京 102206 2北京农学院生物科学与工程学院 北京 102206 3北京农学院农业部都市农业(北方)重点开放实验室 北京 102206
Author(s):
PENG Bo BU Chunya SHI Guanglu LI Jinling FENG Xiaojiao HAN Jingyu GAO Pin WANG Younian
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
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
Keywords:
Tetranychus cinnabarinus acetylcholinesterase (AChE) prokaryotic expression purification half maximal inhibitory concentration (IC50)
分类号:
Q786 : S433.7
DOI:
10.3724/SP.J.1145.2014.08008
文献标志码:
A
摘要:
乙酰胆碱酯酶(AChE)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AChE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pET-30a中,构建表达载体pET-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AChE为抗原制备抗螨AChE特异性抗体;改良的Ellman法测定螨AChE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AChE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AChE特异性抗体. 采用重组AChE(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AChE体外高效表达载体并获得了具有较好活性的重组螨AChE,可为抗AChE杀螨剂的体外高通量筛选和以AChE为靶标的农药残留检测奠定基础.
Abstract:
Acetylcholinesterase (AChE) plays an important role in the process of neural?conduction, and is the major target of 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 (Mr) of 68 × 103 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.

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相似文献/References:

[1]刘洪霞,高希武,冷培恩.花翅摇蚊乙酰胆碱酯酶的底物专一性及对胆碱酯酶抑制剂敏感度的比较[J].应用与环境生物学报,2008,14(02):211.
 LIU Hongxia,et al..Substrate Specificity of Acetylcholinesterase and Its Sensitivity to Cholinesterase Inhibitor in Chironomus kiinensis[J].Chinese Journal of Applied & Environmental Biology,2008,14(02):211.
[2]王霞,刘铭钧,高俊娥,等.太阳鱼脑乙酰胆碱酯酶的分离纯化[J].应用与环境生物学报,2011,17(01):135.[doi:10.3724/SP.J.1145.2011.00135]
 WANG Xia,LIU Mingjun,GAO June & LI Dun.Purification of Acetylcholinerase from Lepomis gibbosus Brain[J].Chinese Journal of Applied & Environmental Biology,2011,17(02):135.[doi:10.3724/SP.J.1145.2011.00135]
[3]冯晓姣,卜春亚,师光禄,等.朱砂叶螨乙酰胆碱酯酶基因的克隆与特征分析[J].应用与环境生物学报,2014,20(05):751.[doi:10.3724/SP.J.1145.2014.01051]
 FENG Xiaojiao,BU Chunya,SHI Guanglu,et al.Gene cloning and character analysis of acetylcholinesterase from Tetranychus cinnabarinus[J].Chinese Journal of Applied & Environmental Biology,2014,20(02):751.[doi:10.3724/SP.J.1145.2014.01051]
[4]彭博,卜春亚,师光禄,等.基于杀螨剂靶标朱砂叶螨乙酰胆碱酯酶体外表达的新型杀螨活性化合物筛选[J].应用与环境生物学报,2016,22(01):110.[doi:10.3724/SP.J.1145.2015.05049]
 PENG Bo,BU Chunya,et al.Screening new acaricidal compound based on In vitro expression of acaricidal target acetylcholinesterase of Tetranychus cinnabarinus[J].Chinese Journal of Applied & Environmental Biology,2016,22(02):110.[doi:10.3724/SP.J.1145.2015.05049]

备注/Memo

备注/Memo:
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 Supported by the National Natural Science Foundation of China (31200483), the Beijing Natural Science Foundation (6122005), the Key Project of Chinese Ministry of Education (212001), the Program of Beijing Municipal Commission of Education (KM201210020001), and the Platform Construction Program of Beijing Municipal Commission of Education (KCT2014001)
更新日期/Last Update: 2015-04-27