论文部分内容阅读
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 prepare TA cloning vector with secreted alkaline phosphatase tracing and eukaryotic expression. Methods The CMV promoter region-TA cloning site-BGH polyadenylation region was cloned into pSEAP2-control vector to construct pcDNA5AP-T vector with eukaryotic expression and exocrine alkaline phosphatase (SEAP) . The enhanced green fluorescent protein (EGFP) gene was cloned into pcDNA5AP-T vector and analyzed for EGFP expression and SEAP activity. Results The pcDNA5AP-EGFP was transfected into 6-well 293T cells at 400ng / well, and the EGFP expression rate was 50%. The SEAP activity was 20 times higher than that of the control group. Conclusion The pcDNA5AP-T vector with eukaryotic expression and exocrine alkaline phosphatase was successfully constructed.