分泌型碱性磷酸酶示踪的TA克隆真核表达载体的构建

Construction of secreted alkaline phosphatase traced and eukaryotic expressed TA cloning vector

目的制备具有分泌型碱性磷酸酶示踪作用和真核表达功能的TA克隆载体。方法把CMV启动子区-TA克隆位点-BGH多聚腺苷酸区序列克隆到pSEAP2-control载体中,构建成具有真核表达功能并外分泌碱性磷酸酶(SEAP)的pcDNA5AP-前T载体。增强型绿色荧光蛋白(EGFP)基因克隆到pcDNA5AP-T载体上并进行EGFP表达分析和SEAP活性检测。结果 pcDNA5AP-EGFP以400ng/孔转染6孔板293T细胞,EGFP表达率达50%,SEAP活性比对照组高20倍。结论成功构建了具有真核表达功能并外分泌碱性磷酸酶的pcDNA5AP-T载体。

Objective To construct an eukaryotic expressed TA cloning vector that is traced by secreted alkaline phosphatase（SEAP）.Methods To construct a secreted alkaline phosphatase traced and eukaryotic expressed pre-T vector of pcDNA5AP,a sequence that contains a CMV promoter and a TA cloning site was cloned into pSEAP2-control vector,the EGFP gene was cloned into pcDNA5AP-T vector to confirm the efficacy of this vector.The level of EGFP and SEAP was analysed.Results Transfected with 400ng pcDNA5AP-EGFP vector in 3cm well,50% of 293T cells could be deteced of EGFP expression,the corresponding SEAP value in supernatant was 20 times higher than negtive control.Conclusions Eukaryotic expressed TA cloning vector that is traced by secreted alkaline phosphatase （pcDNA5AP-T vector） is constructed successfully.