| 摘要: |
| 【目的】在昆虫草地贪夜蛾细胞（Sf9）中表达一种能专一阻断昆虫钠、钾离子通道的神经毒素基因RjAa17f。【方法】运用杆状病毒真核表达系统表达RjAa17f毒蛋白，并用蛋白质印迹对其进行检测；测定RjAa17f毒蛋白引起健康棉铃虫中肠膜电位的变化，并对其毒力进行测定。【结果】通过重组杆状病毒质粒的转染和Western blot检测验证，神经毒素基因RjAa17f的重组Bacmid已经成功转染到Sf9细胞中，且在Sf9中表达出与预测大小一致的RjAa17f目的蛋白；此毒蛋白作用于棉铃虫中肠3～12 h后，中肠膜电位与CK相比差异显著，且随着作用时间的延长，棉铃虫中肠膜电位呈逐步升高的趋势，作用9 h以后，升高的幅度逐渐降低，趋于平缓。RjAa17f毒蛋白对棉铃虫的LD50为108.12 μg/g。【结论】在Sf9细胞内表达出了有活性的RjAa17f毒蛋白，且该毒蛋白对棉铃虫中肠膜电位有显著改变。 |
| 关键词: 棉铃虫 神经毒素基因RjAa17f Sf9 RjAa17f毒蛋白 杆状病毒真核表达系统 |
| DOI： |
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| 基金项目:国家自然科学基金项目（31170609，31270691） |
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| Expression and activity of neurotoxin gene RjAa17f in Sf9 cells |
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MENG Jiao,YU Huan,ZHOU Bin,et al
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| Abstract: |
| 【Objective】This study aimed to express Rhopalurus junceus neurotoxin gene in Spodoptera frugiperda cells (Sf9) to block insect sodium and potassium ion channels.【Method】The toxic protein RjAa17f was expressed by baculovirus eukaryotic expression vector system,and western blots was used to identify the toxic protein.The change of midgut membrane potential caused by RjAa17f toxic protein crude extract was determined and the toxicity was tested.【Result】By Western blot verification,RjAa17f target protein expressed in Sf9 had the same size as predicted.After acting on Helicoverpa armigera midgut,the midgut membrane potential increased significantly during 3-12 h compared to CK and the trend decreased after 9 h.LD50 of RjAa17f toxin protein crude extract was 108.12 μg/g.【Conclusion】RjAa17f active protein was expressed in Sf9,and it significantly changed midgut potential of Helicoverpa armigera. |
| Key words: Helicoverpa armigera neurotoxin gene RjAa17f Sf9 toxic protein RjAa17f baculovirus eukaryotic expression system |
