朱砂叶螨乙酰胆碱酯酶真核表达载体构建及在293T细胞中表达条件

Construction eukaryotic expression vector of Tetranychus cinnabarinus acetylcholinesterase and its expression optimization in 293T cell

乙酰胆碱酯酶(AChE;EC 3.1.1.7)是神经传导中调节神经递质乙酰胆碱水平的一种关键酶,是有机磷和氨基甲酸酯类农药的主要作用靶标。目前关于朱砂叶螨乙酰胆碱酯酶的真核表达还未见报道。本研究拟构建朱砂叶螨AChE的真核表达载体,进行在293T细胞中表达条件摸索。将朱砂叶螨野生型ace完整读框和去掉3’疏水区的ace基因分别插入到真核表达载体pcDNA3.1/V5-His-TOPO中,经PCR和双酶切鉴定及测序验证后,成功构建了真核表达载体pcDNA3.1/ace。采用GFP报告基因确定在293T细胞中表达条件摸索,结果表明293T细胞的最适转染时间为48h和LipofectamineTM2000转染试剂的最适加样量为20μL。本试验成功构建了朱砂叶螨AChE的真核表达载体,确定了在293T细胞中的表达条件,为后续筛选新型选择性AChE抑制剂奠定了基础。

Acetylcholinesterase(AChE)plays a key role in normal nerve impulse transmission by controlling ACh level,and it is the main target of organophosphate and carbamate insecticides.The expression of mite AChE in eukaryotic cell is not reported yet.The aim of this study is to construct eukaryotic expression vector of Tetranychus cinnabarinus ace and optimize its expression in 293 Tcell.The ace code region and truncated ace gene were respectively inserted into expression vector pcDNA3.1/V5-His-TOPO,which was verified by PCR,double enzyme digestion and sequencing.GFP report gene was used to optimize the transfection addition level and the time to collect 293 Tcell after transfection.The results showed that highest expression efficiency was obtained when addition of 20μL LipofectamineTM2000 in 6cm culture dish at 48 hpost transfection.The constructed T.cinnabarinus AChE expression vector with its expression conditions optimized laid a solid foundation for screening new selective acetylcholinesterase inhibitors.