摘要: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cDN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cDN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。
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