Carboxylesterases 在抗杀虫剂家蝇、家蝇中的功能表征
Summary
在这里, 我们提出了一个协议, 生产的房子飞羧酸蛋白的体外与杆状病毒介导的昆虫细胞表达系统, 后来功能表征其在代谢氯菊酯的作用, 从而, 赋予以传导细胞为基础的 mtt 法和体外代谢研究的抗性。
Abstract
羧酸介导的代谢在各种昆虫的抗药性中起着重要的作用。在耐药宫蝇株中发现了几种羧酸基因, 而它们在赋予杀虫剂抗药性方面的作用仍有待探讨。在这里, 我们设计了一个 carboxylesterases 功能特性的协议。三例实验表明: (1) 羧酸蛋白的表达和分离, 通过杆状病毒介导的昆虫斜纹 frugiperda (Sf9) 细胞表达系统;(2) 以细胞为基础的 MTT 法 (3-[4, 5-dimethykthiazol 2-基] 2, 5-diphenyltetrazolium 溴化物) 细胞毒性试验, 测定不同氯菊酯处理对昆虫细胞的耐受性;(3)体外代谢研究, 探讨 carboxylesterases 对氯菊酯的代谢能力。羧酸基因 MdαE7 是克隆自抗性家飞应变 ALHF, 并用于构建重组杆状病毒 Sf9 细胞感染。用 MTT 法测定了不同氯菊酯治疗可行性细胞的含量。实验组 (MdαE7-recombinant 杆状病毒感染细胞) 的增强耐受性与对照组 (CAT 重组杆状病毒感染细胞和 GFP-重组杆状病毒感染细胞) 相比, 对氯菊酯治疗建议的能力, MdαE7 在代谢杀虫剂, 从而保护细胞免受化学损害。此外, 羧酸蛋白在昆虫 Sf9 细胞中表达, 并被分离出来进行体外代谢研究。结果表明, MdαE7 对氯菊酯的体外代谢效率显著, 直接表明 carboxylesterases 参与了杀虫剂的代谢, 从而在室内蝇类中具有抗药性。
Introduction
杀虫剂抗药性目前是一个主要问题的房子飞行控制全球1,2。确定杀虫剂抗药性机制的努力有助于更好地了解这一问题, 从而提供了新的战略, 有效地防止或尽量减少抗药性发展3的蔓延。Carboxylesterases 作为一种主要的解毒酶, 在各种昆虫4、5、6中对杀虫剂的螯合和代谢中的作用引起了人们的极大关注。我们以前的研究已经确定了多 carboxylesterases 的房子苍蝇和他们的表达水平不仅组成性在抗 ALHF 菌株, 但也可以诱导到较高水平的反应氯菊酯治疗7.然而, 这些羧酸基因在代谢杀虫剂中的功能特征仍有待探讨。
自从8二十世纪八十年代初的第一次报告以来, 由于其高蛋白的生产效率和真核蛋白处理能力9, 一种杆状病毒介导的国外基因表达系统得到了广泛的应用。该二元体系由两个基本要素组成: 构建的重组杆状病毒将外来基因传递给宿主细胞, 以及由重组杆状病毒感染的细胞大规模表达感兴趣的蛋白质。在过去的几十年中, 杆状病毒介导的细胞表达系统已被广泛用于生产数以千计的重组蛋白, 从胞浆酶到膜结合蛋白的昆虫和哺乳动物细胞10。我们以前的研究成功地表达了昆虫 Sf9 细胞中的多种 CYP450 酶, 这个系统11。在本研究中, 我们构建了羧酸重组杆状病毒感染昆虫 Sf9 细胞, 探讨了细胞对不同氯菊酯治疗的耐受性, 以及大尺度表达的羧酸蛋白在体外功能探索。而不是从昆虫组织匀浆的多种羧酸同工酶混合物的研究12,13, 这种杆状病毒介导的昆虫细胞表达系统允许特定的表达和分离靶蛋白, 更好地表征其生物化学和结构特性。
四氮唑盐基检测 (MTT) 是一种高通量比色法, 用于测量细胞活力。这一分析的基础上, 只有活细胞能够代谢黄色的 MTT 试剂到深紫色的彩色 formazan 沉淀, 可以 colorimetrically 分析后溶解在有机溶剂14, 15。近年来, 一些比较准确但耗时的方法, 如台盼蓝排斥和胸苷滴定法16,17。然而, 基于细胞的 mtt 检测目前仍然被认为是最快速和最容易操作的方法, 快速检测细胞的生存能力。在这里, 我们使用 MTT 法来探讨细胞对杀虫剂治疗的耐受性。羧酸重组杆状病毒感染后细胞的增强耐受性强烈支持 carboxylesterases 对杀虫剂的代谢作用, 这反过来又表明它们参与了杀虫剂抗药性的研究。
此外, 本研究还进行了体外代谢试验。与一般的羧酸, 如α-napthyl 醋酸酯 (α钠) 和β萘乙酸 (β钠) 共同基质, 以反映 carboxylesterases 的水解活动,体外代谢研究被认为是一个准确的方法直接测量 carboxylesterases 对杀虫剂的活动18。该方法已成功地应用于各种昆虫, 以表征多种细胞色素 P450s 与杀虫剂抗药性11,19,20。然而, 这种方法尚未在羧酸研究中得到应用。利用杆状病毒介导的表达系统所产生的羧酸蛋白, 可以对氯菊酯 carboxylesterases 进行体外代谢研究, 进一步提供有力的证据证明参与carboxylesterases 在给拟除虫菊酯抗性的房子苍蝇。
Protocol
Representative Results
Discussion
近几十年来, 异基因表达系统被广泛用于表达和分离大量的蛋白质, 从而允许生化和功能的测定和酶的体外鉴定。到目前为止, 几种不同的模型系统, 包括大肠杆菌,毕赤酵母, Sacccharomyces 酵母和斜纹夜蛾 frugiperda已被改编为重组蛋白表达, 并选择体外系统是重要的大规模产生感兴趣的蛋白质22,23,…
Materials
| Q5 High-Fidelity DNA Polymerase | New England Biolabs inc. | M0491L | |
| QIAquick Gel Extraction Kit | QIAGEN | 28704 | |
| pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli | Invitrogen by life technology | K240020 | S.O.C medium and universal M13 sequence primers were included in this kit. |
| PureLink HiPure Plasmid Miniprep Kit | Invitrogen by life technology | K210002 | |
| Gateway LR Clonase II Enzyme mix for BaculoDirectTM Kits | Invitrogen by life technology | 11791-023 | |
| BaculoDirect C-Term Linear DNA Transfection Kit | Invitrogen by life technology | 12562-019 | Cellfectin transfection reagent and ganciclovir were included in this kit |
| pENTR-CAT plasmid | Invitrogen by life technology | Included in BaculoDirect C-Term Linear DNA Transfection Kit, concentration: 0.5 ug/uL | |
| Heat inactivated Fetal Bovine Serum, Certified | Gibco by Life Technologies | 10082-139 | |
| Sf9 cells in Sf-900 III SFM | Gibco by Life Technologies | 12659017 | |
| Insect Cell-PE LB Insect Cell Protein Extraction & Lysis Buffer | G Biosciences by A Geno Technology Inc | 786-411 | |
| Sf-900 III SFM (1×) Serum Free Medium Complete | Gibco by Life Technologies | 12658-019 | |
| Grace's Insect Medium, unsupplemented | Gibco by Life Technologies | 11595030 | |
| Permethrin (isomers) analytical standard | SUPELCO by Solutions WithinTM | 442748 | |
| Methanol (analytical graded) | Sigma-Aldrich | 67-56-1 | |
| Acetonitrile (analytical graded) | Sigma-Aldrich | 75-05-8 | |
| GHP Acrodisc 25 mm Syringe Filters with 0.45 μm GHP Membrane (HPLC Certified) | Pall Life Sciences | 21890388 | |
| Alliance Waters 2695 HPLC System | Waters | ||
| T100 Thermal Cycle | Bio-Rad Laboratories Inc. | 1861096 | |
| Nanodrop 2000/2000c Spectrophotometers | ThermoFisher Scientific | ND2000CLAPTOP | |
| Cytation 5 Cell Imaging Multi-Mode Reader | BioTek |
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