摘要
目的:构建同时沉默Livin基因和MTA1基因的小干扰RNA(siRNA)载体。方法:设计Livin和MTA1的siRNA靶序列,合成它们互补的寡核苷酸链,分别退火后重组入pRNAT-U6.2载体,得到pRNAT-U6.2-Livin和pR-NAT-U6.2-MTA1。AccⅡ和XhoⅠ酶切pRNAT-U6.2-MTA1,回收475bp的MTA1siRNA单元,然后与XhoⅠ和PmeⅠ酶切线形化的pRNAT-U6.2-Livin重组,得到pRNAT-U6.2-Livin-MTA1,后者用脂质体包被转染人食管癌细胞EC9706,采用RT-PCR法分别检测转染细胞Livin和MTA1基因mRNA的表达情况。结果:针对Livin基因和MTA1基因的siRNA的双链寡核苷酸片段分别克隆入pRNAT-U6.2载体,经测序分析插入片段正确。PCR扩增证实MTA1siRNA单元重组入pRNAT-U6.2-Livin。RT-PCR检测显示,转染pRNAT-U6.2-Livin-MTA1的EC9706细胞Livin基因和MTA1基因mRNA表达水平皆明显降低。结论:同时沉默Livin基因和MTA1基因表达的siRNA载体构建成功。
Aim: To construct the siRNA expression vector targeting Livin and MTA1. Methods:The siRNA target sequence of Livin and MTA1 were designed, complementary oligonucleotide sequences were synthesized. The annealed oligonucleotide fragments were cloned into pRNAT-U6.2 expression vector, pRNAT-U6.2-Livin and pRNAT-U6.2-MTA1 were obtained, pRNAT-U6.2-MTA1 was digested with Acc Ⅱ and Xho Ⅰ , 475 bp MTA1 siRNA fragment was retrieved, then recombinated with Xho Ⅰ/Pme Ⅰ digested pRNAT-U6.2-Livin, finally pRNAT-U6.2-Livin-MTA1 was gained. The recombinant plasmids were transfected into EC9706 cells wrapped with liposome. The mRNA expression of Livin and MTA1 in the stable transfected cells was assayed by RT-PCR. Results: siRNA double strands oligonucleotide fragments aimed at Livin and MTA1 were cloned into pRNAT-U6.2 vector, DNA sequencing showed that the insertion elements were correct. PCR amplification confirmed that MTA1 siRNA element was recombinated with pRNAT-U6.2-Livin. RT-PCR result showed that Livin and MTA1 mRNA expression in the EC9706 cells transfected pRNAT-U6.2-Livin-MTA1 was down-regulated. Conclusion: The siRNA expression vector targeting Livin and MTA1 was successfully constructed.
出处
《郑州大学学报(医学版)》
CAS
北大核心
2008年第2期202-205,共4页
Journal of Zhengzhou University(Medical Sciences)
基金
教育部“十五”项目
教育部“211”工程重点学科建设基金资助项目教重办2002第2号