特异性沉默Eca109细胞系stathmin基因siRNA表达载体构建.docVIP

特异性沉默Eca109细胞系stathmin基因siRNA表达载体构建 　 作者：王峰，王留兴，王瑞林，樊青霞，赵培荣 【摘要】 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stathmin mRNA的表达。结果重组逆转录病毒载体经EcoRⅠ、Hind Ⅲ双酶切，可见4692 nt和285 nt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。 【关键词】 小干扰RNA；stathmin基因；pSUPEREGFP载体；Eca109细胞 　　Abstract：Objective To construct and identify a recombinant retroviral vector pSUPERS that target stathmin gene and a stable virusproducing cell line.MethodsThe 64nt encoded targeting stathmin gene shRNA sequence was cloned into a retroviral vector pSUPEREGFP with DNA recombinant technique. The recombinant vector was identified by the electrophoresis analysis of restriction 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 transfected Eca109 cell lines.ResultsThe electrophoresis of EcoRⅠand HindⅢ digested products showed two DNA fragments, 4692 nt and 285 nt, respectively. The result of sequence demonstrated 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 treated groups with pSUPERS.ConclusionA recombinant retroviral vector pSUPERS that specific silence stathmin gene and a stable virusproducing packaging cell line were successfully constructed and identified. 　　Key words：Small interfering RNA; stathmin gene; pSUPEREGFP vector; Eca109 cells 　　0引言 　　食管癌是常见的恶性肿瘤之一。某些基因的异常表达与肿瘤发生、发展密切相关。Stathmin为一种高度保守的细胞内蛋白，在多种恶性肿瘤细胞中高表达，研究表明[1,2] 通过应用反义核酸、单克隆抗体、核酶、stathmin蛋白抑制