人类Survivin基因真核表达载体的构建及其在K562细胞中的表达

 目的　构建带有绿色荧光蛋白的Survivin基因真核表达载体pIRES2-EGFP／Survivin，并转染K562细胞。方法　以 pDNR／Survivin质粒为模板，PCR扩增Survivin基因，T-A 克隆，亚克隆至pIRES2-EGFP上，并对重组表达载体进行酶切、测序鉴定。采用Superfect转染试剂转染K562细胞。提取细胞RNA，RT-PCR检测Survivin基因mRNA表达。结果　经限制性酶切鉴定及测序分析证实pIRES2-EGFP／Survivin载体序列正确；荧光显微镜下可见转染的K562细胞有绿色荧光蛋白的表达，RT-PCR证实转染细胞中存在Survivin基因mRNA的表达。结论　pIRES2-EGFP／Survivin表达载体构建成功，并在K562细胞内成功表达。

Construction of human eukaryotic vector with expression of Survivin in K562 cells

Objective To construct an eukaryotic vector with expression of human survivin gene with green fluorescent protein which is named pIRES2-EGFP/survivin and transfected into K562 cell line.Methods Using pDNR/Survivin plasmid as a template, the full length of survivin cDNA was amplified by PCR and subsequently cloned into T-A vector and then subcloned into pIRES2-EGFP vector. After identified by digestion of restrictive endonucleases, pIRES2-EGFP/survivin was further confirmed by sequencing. Then it was transfected into K562 cells with superfect reagents. The mRNA was isolated and survivin gene was detected by Western blotting. Results The exact sequences of pIRES2-EGFP/survivin vector were confirmed by digestion of restrictive endonucleases and sequencing. After transfection, the expressions of green fluorescent protein were present. The mRNA expression of survivin has been detected in transfected cells by RT-PCR. Conclusion The vector pIRES2-EGFP/survivin has been constructed and could express survivin gone in K562 cells successfully.