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中国农学通报

中国农学通报 ›› 2025, Vol. 41 ›› Issue (32): 109-115.doi: 10.11924/j.issn.1000-6850.casb2024-0637

• 植物保护 • 上一篇    下一篇

家蝇乙酰胆碱酯酶在杆状病毒介导的Sf9细胞中表达

伍全根1(), 张秀娟2, 孙英健3()   

  1. 1 黄山学院生命与环境科学学院，安徽黄山 245041
    2 中国科学院动物研究所，北京 100101
    3 北京农学院动物科学与技术学院，北京 102206
  • 收稿日期:2024-10-14 修回日期:2024-12-15 出版日期:2025-11-18 发布日期:2025-11-18
  • 通讯作者:
    孙英健，女，教授，博士，研究方向：药理学与环境毒理学。通信地址：102206 北京市昌平区史各庄街道北农路7号 北京农学院动物科学技术学院，Tel：010-80795592，E-mail：。
  • 作者简介:

    伍全根，男，讲师，本科，研究方向：生物技术。通信地址：245041 安徽黄山市屯溪区西海路39号 黄山学院生命与环境科学学院，Tel：0559-2546631，E-mail：。

  • 基金资助:
    北京市自然科学基金“芍药苷与抗生素联用抗MRSA菌株感染及降低抗生素耐药性的机制研究”(6212003); 北京农学院科技创新“火花行动”支持计划“减抗背景下基于赤芍的抗菌新药的研制与药效研究”(BUA-HHXD2024004)

Expression of Housefly Acetylcholinesterase in Baculovirus-Mediated Sf9 Cells

WU Quangen1(), ZHANG Xiujuan2, SUN Yingjian3()   

  1. 1 College of Life and Environment Sciences, Huangshan University, Huangshan, Anhui 245041
    2 Institute of Zoology, Chinese Academy of Sciences, Beijing 100101
    3 College of Veterinary Medicine and Animal Science, Beijing University of Agriculture, Beijing 102206
  • Received:2024-10-14 Revised:2024-12-15 Published:2025-11-18 Online:2025-11-18

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摘要： 本研究旨在利用基因重组技术与杆状病毒介导的Sf9昆虫细胞表达系统，实现家蝇乙酰胆碱酯酶(AChE)的活性表达，为将其开发为杀虫剂残留检测试剂及昆虫抗药性分析提供基础。以含家蝇AChE基因(Md-ache)的pcDNA3.1-Md-ache质粒为模板，通过PCR扩增引入EcoRⅠ和XhoⅠ酶切位点，构建重组质粒Peasy-T1-Md-ache，进一步与pFastBac1载体连接获得重组质粒pFastBac1-Md-ache，转染DH10Bac感受态细胞后筛选得到重组bacmid质粒，再转染Sf9细胞进行蛋白表达与鉴定。结果表明：（1）重组bacmid质粒在Sf9细胞中成功包装，转染后细胞出现体积增大、增殖抑制等典型病变；（2）重组家蝇AChE具有催化活性，细胞裂解液中酶活性达44.7±2.3 nmol/(min·mg protein)，培养液中检测到10.1±2.1 nmol/(min·mL)的酶活性，提示存在分泌性表达；（3）经Lineweaver-Burk双倒数作图法测定，重组AChE的Km值为15.69±1.75 µmol/L，对底物亲和力较高；（4）有机磷杀虫剂毒死蜱可体外抑制该重组AChE活性，经改良寇氏法计算半数抑制浓度(IC50)为11.18±0.68 µmol/L。本研究证实，杆状病毒介导的Sf9细胞表达系统可高效表达具有催化活性与杀虫剂敏感性的家蝇AChE，其表达产物为杀虫剂残留检测试剂开发及昆虫抗药性机理分析提供了关键材料。

关键词: 家蝇, 乙酰胆碱酯酶, ache基因, 基因重组, 杆状病毒表达系统, Sf9细胞, 有机磷杀虫剂, 氨基甲酸酯杀虫剂

Abstract:

This study aims to express housefly acetylcholinesterase (AChE) using gene recombination technology and a baculovirus-mediated Sf9 insect cell expression system, to provide a scientific basis for the development of AChE as a reagent for insecticide residue detection and insect resistance analysis. Using the pcDNA3.1-Md-ache plasmid containing the housefly AChE gene (Md-ache) as a template, EcoRⅠ and XhoⅠ restriction sites were introduced through PCR amplification to construct the recombinant plasmid Peasy-T1-Md-ache, which was further ligated with the pFastBac1 vector to obtain the recombinant plasmid pFastBac1-Md-ache. After transfection into DH10Bac competent cells, recombinant bacmid plasmids were screened and subsequently transfected into Sf9 cells for protein expression and identification. The results demonstrated that (1) the recombinant bacmid plasmids were successfully expressed in the Sf9 cells, with transfected cells exhibiting typical cytotoxic effects including cell enlargement and proliferation inhibition; (2) the recombinant housefly AChE exhibited catalytic activity, with enzyme activity reaching 44.7 ± 2.3 nmol/(min·mg protein) in cell lysates and 10.1 ± 2.1 nmol/(min·mL) in the culture medium, indicating secretory expression; (3) the Km value of the recombinant AChE was measured to be 15.69 ± 1.75 μmol/L using the Lineweaver-Burk double-reciprocal plot method, suggesting high substrate affinity; (4) the organophosphorus insecticide chlorpyrifos inhibited the recombinant AChE activity in vitro, with a half-maximal inhibitory concentration (IC50) of 11.18 ± 0.68 μmol/L calculated by using the modified Karber method. This study confirms that the baculovirus-mediated Sf9 cell expression system can efficiently express housefly AChE with both catalytic activity and sensitivity to insecticide. The expression product provides a key material for the development of the reagent which is expected to be used for the insecticide residue detection and the insect resistance analysis.

Key words: housefly, acetylcholinesterase, AChE gene, gene recombination, baculovirus expression system, Sf9 cell, organophosphorus insecticide, carbamate insecticide