绿色荧光蛋白标记的NTE活性域表达载体的构建及表达

Construction and expression of eukaryotic expression vector of human neuropathy target esterase domain tagged with green fluorescence protein

利用含有特定限制性内切酶识别位点的引物,通过聚合酶链式反应扩增出人神经病靶标酯酶活性域的编码序列,经T载体克隆测序正确后,双酶切回收特异片段定向插入到增强型绿色荧光表达载体pEGFP-N3中,通过酶切反应鉴定,构建了绿色荧光蛋白标记的神经病靶标酯酶活性域的融合表达载体pNEST-EGFP。采用脂质体转染的方法将其转染到人神经瘤母瘤细胞SH-SY5Y中,用荧光显微镜观察发现神经病靶标酯酶活性域分布于细胞质中,而且没有导致内质网膜的聚集,表明表达载体成功构建和表达。

The eDNA sequence of human neuropathy target esterase （NTE） esterase domain （NEST） was amplified by polymerase chain reaction （PCR） with special primers including specific restrict endocleavease site and then cloned into T vector. After the recombinant plasmid was certified by DNA sequencing, the eDNA fragments were inserted into eukaryotic expression vector pEGFP-N3 to generate the eukaryotic expression vector of NEST tagged with enhenced green fluorescence protein （EGFP）, pNEST, which was identified with digestion of restrict endocleavease. Then pNEST EGFP was transiently trans-infected into human neuroblastoma SHSY5Y cells by liposome methods and detected with fluorescence microscope. NEST tagged with EGFP was distributed cytoplasm and did not induce aggregation of endoplasmic reticulum membrane, which indicates that eukaryotic expression vector of NTE gene esterase activity domain has been constructed successfully and expressed efficiently.