特异性沉默Eca109 细胞系stat hmin 基因siRNA 表达载体的构建

 目的 构建并筛选特异性沉默stathmin基因的pSUPER-S逆转录病毒载体以及稳定表达的细胞克隆。方法 用DNA重组技术,将64nt能转录产生靶向stathmin小发夹RNA（shRNA）的寡核苷酸序列,定向克隆入逆转录病毒载体pSUPER-EGFP,应用脂质体将重组逆转录病毒载体pSUPER-S转染细胞系Eca109,G418筛选建立稳定产生逆转录病毒的细胞克隆,RT-PCR检测转染细胞stathminmR-NA的表达。结果 重组逆转录病毒载体经EcoRⅠ、HindⅢ双酶切,可见4692nt和285nt条带;测序结果表明插入序列正确;重组载体转染Eca109细胞,可表达绿色荧光蛋白（EGFP）,经G418筛选得到抗性细胞克隆,转染细胞stathmin mRNA的表达较对照组明显减弱。结论 特异性沉默stathmin基因的pSUPER-S逆转录病毒载体以及稳定表达的细胞系构建和筛选成功。

Construction of Specific Silencing Vector-based siRNA on stathmin Gene Expression in Eca109 Cell Line

Objective 　To construct and identify a recombinant retroviral vector pSUPER-S that target stathmin gene and a stable virus-producing cell line. Methods 　The 64nt encoded targeting stathmin gene shRNA sequence was cloned into a ret roviral vector pSUPER-EGFP with DNA recombinant technique.The recombinant vector was identified by the electrophoresis analysis of rest riction enzyme digestion and DNA sequencing. The packaging cell Eca109 was transfected with this recombinant plasmid using liposome-based transfection and the stable intergrant was selected by using G418 medium. The expression of stathmin mRNA was detected by RT-PCR in t ransfected Eca109 cell lines. Results 　The elect rophoresis of EcoR Ⅰand Hind Ⅲdigested product s showed two DNA fragments , 4692 nt and 285 nt , respectively.The result of sequence demonst rated that 64nt had been inserted into the vector. Green fluorescent protein ( EGFP) was observed after transfection. G418 resistant clones were also selected. Comparing with control groups the expression of stathmin gene was inhibited obviously in t reated group s with pSUPER-S. Conclusion 　A recombinant retroviral vector pSUPER-S that specific silence stathmin gene and a stable virus-producing packaging cell line were successfully constructed and identified.