螨ace/S154G突变型原核表达载体的构建及其蛋白纯化

Constructing prokaryotic expression vector of acetylcholinesterase mutant ace/S154G from Tetranychus cinnabarinus and its protein purification

【目的】体外表达朱砂叶螨乙酰胆碱酯酶突变型ace/S154G来探究世界性害螨AChE S154位点的功能。【方法】构建pET-30a/ace/S154G重组表达载体,诱导表达后纯化目的蛋白,并进行Western Blot检测。【结果】ace/S154G在pET-30a载体上成功高水平表达,对AChE/S154G进行大量表达和纯化,经Western Blot验证,获得纯度较高的目的蛋白。【结论】实现ace/S154G体外高效原核表达,获得AChE/S154G突变体重组蛋白,为探明朱砂叶螨AChE的S154位点的功能,研发新型选择性杀螨剂奠定基础。

【Objective】In vitro expression of acetylcholinesterase mutant ace/S154G of Tetranychus cinnabarinus were used to determine the function of AChE site S154 of worldwide pest mite.【Methods】The pET-30a/ace/S154G recombinant expression vector was constructed and expressed in E.coli.The target protein was purified and detected by Western Blot.【Results】ace/S154G mutant was successfully expressed at high level in pET-30a vector.High quality of AChE/S154G mutant protein were purified,and was verified by Western Blot.【Conclusion】In this study,ace/S154G mutant was efficiently expressed in prokaryotic cells,and high quality of AChE/S154G mutant proteins were obtained,which laid the foundation for determining the function of spider mite acetylcholinesterase S154 site and development of new selective acaricides.