NTE酯酶活力域真核表达载体的构建及其表达

目的构建人神经病变靶标酯酶（NTE）酯酶活力域（NESP）真核表达载体，并在细胞中表达。方法利用含有特定酶切位点的引物，通过PCR扩增出其酯酶活力域，经T载体克隆测序正确后，双酶切回收cDNA片段定向插入到pcDNA3．1（＋）中，通过酶切鉴定其真核表达载体的构建。然后将其转染到真核细胞中，通过NTE活力测定，检测其表达效果。结果经PCR获得了NTE的1．6kb酯酶活力域的cDNA片段，T载体克隆后测序证实完全正确，然后克隆到真核表达载体中获得了NTE酯酶活力域真核表达载体pcDNA—NEST。瞬时转染到COS7和SH-SY5Y细胞中，NTE的活力与对照细胞相比显著提高。结论成功构建了NEST真核表达载体，并在哺乳动物细胞中表达。

Construction of a eukaryotic expression vector for expression of esterase domain of human neuropathy target esterase

Objective To construct a eukaryotic expression vector for expression of human neuropathy target esterase(NTE) esterase domain in mammalian cells.Method The cDNA fragment of NTE esterase domain (NEST) was amplified by PCR with special primers including specific restrict endocleavease site and then cloned into pGEM-T easy vector. After the recombinant plasmid were certified by DNA sequencing, the cDNA fragments were inserted into eukaryotic expression vector pcDNA3.1 (+) to generate a vector (named as pcDNA-NEST) for expression of NEST and the constructed vector was confirmed with restrict endocleaveases, pcDNA-NTE was transiently transfected into COS7 and SH-SY5Y cells and the activities of NTE in the cells were assayed.Results A 1.6 kb fragment of NTE esterase activity domain was obtained by PCR. After cloned by pGEM-T easy vector and confirmed by DNA sequencing,eukaryotic expression vector of NTE gene esterase activity domain, pcDNA-NEST was successfully constructed by using recombinant DNA technique. The activity of NTE in the mammalian cells was increased significantly after transfection of pcDNA-NEST.Conclusion Eukaryotic expression vector of NTE gene esterase activity domain has been constructed successfully and expressed efficiently in the mammalian cells.