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董双林, 韩召军. 2006: 利用Bac-to-Bac系统表达棉蚜的乙酰胆碱酯酶. 动物学研究, 27(1): 75-80.
引用本文: 董双林, 韩召军. 2006: 利用Bac-to-Bac系统表达棉蚜的乙酰胆碱酯酶. 动物学研究, 27(1): 75-80.
DONG Shuang-lin HAN Zhao-jun Martin S WILLIAMSON , *. 2006. Expression of the Aphis gossypii AChE1 Gene Using the Baculovirus Expression System. Zoological Research, 27(1): 75-80.
Citation: DONG Shuang-lin HAN Zhao-jun Martin S WILLIAMSON , *. 2006. Expression of the Aphis gossypii AChE1 Gene Using the Baculovirus Expression System. Zoological Research, 27(1): 75-80.

利用Bac-to-Bac系统表达棉蚜的乙酰胆碱酯酶

Expression of the Aphis gossypii AChE1 Gene Using the Baculovirus Expression System

  • 摘要: Bac-to-Bac昆虫杆状病毒表达系统是一种高效真核表达系统。采用重组DNA技术构建棉蚜乙酰胆碱酯酶1(AChE1)表达质粒,应用该系统在昆虫Sf9细胞系中成功表达了棉蚜的AChE1。表达的AChE以可溶性形式释放于细胞培养液中,对其自然底物类似物ATChI的Km值为(144.5±28.6)μm;对抗蚜威、唑蚜威、氧化乐果和灭赐松等4种杀虫剂的敏感性指数(Ki值)分别为513、1 883、5和23(mM·min) -1,与文献报道的测定结果基本一致。昆虫细胞被重组病毒感染后第2~3天所得产物的活性最高;该产物在-20 ℃保存90 d后活性没有明显下降,4 ℃保存60 d后下降40%,室温(22 ℃)保存7 d后即下降70%。

     

    Abstract: Using the Bac-to-Bac baculovirus expression system, the acetylcholinesterase 1 gene (ace1) of Aphis gossypii was successfully expressed in Sf9 cells. The enzyme was secreted into the cell culture medium and tested. The affinity (Km) of the expressed AChE1 for the substrate analog ATChI was 144.5±28.6 μM. The sensitivity index (indicated by Ki value) of expressed AChE1 to 2 carbamate insecticides (pirimicarb and triazamate) and 2 organophosphate insecticides (omethoate and demeton-S-methyl) were 513, 1 883, 5 and 23 (mM·min) -1, respectively, which is in agreement with previously reported values obtained with the natural enzyme. The expressed enzyme showed highest activity when harvested at 2-3 d after infection of the insect cells. The expressed AChE1 was very stable without obvious loss in enzyme activity after 90 d when stored at -20 ℃, although 70% of the enzyme activity was lost after 7 d when stored at room temperature.

     

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