SiRNA真核表达载体阻断HeLa细胞stathmin基因表达研究

Blocking effect of vector-based siRNA on stathmin gene expression in human HeLa cells

目的构建人stathmin基因的siRNA真核表达载体,探讨其对HeLa细胞中stathmin基因表达的干涉作用.方法将合成的siRNA寡核苷酸链退火形成双链,连接入经BamHⅠ和HindⅢ双酶切后的pSilencer4.1-CMV neo真核表达载体,酶切及测序鉴定.脂质体法转染重组质粒入HeLa细胞,G418筛选后RT-PCR检测其对stathmin基因mRNA的干涉效果.结果经酶切及测序鉴定,成功构建siRNA真核表达载体.经脂质体转染HeLa细胞后,RT-PCR显示所构建的干涉stathmin基因的真核表达载体成功地抑制了目的基因的表达,HeLa细胞中stathmin基因的mRNA表达水平明显降低.结论成功构建了人stathmin基因的RNA干涉真核表达载体pSilencer-S1和pSilencer-S2,并在HeLa细胞中有效地发挥了对stathmin基因表达的干涉作用.

AIM： To explore the blocking effect of siRNA on the expression of stathmin gene in HeLa cell line using siRNA eukaryotic expression vector. METHODS; Two stathmin siRNA cDNAs were synthesized according to the stathmin gene sequence and cloned into the vector pSilencer4.1-CMV-neo and named pSilencer-S1 and pSilencer-S2 respectively, which were further identified by restriction endonuclease digestion analysis and DNA sequencing. HeLa cells were then transfected with pSilencer-S1 and pSilencer- S2. After G418 selection, the cells were selected and the interfering effect was detected by RT-PCR. RESULTS. Restriction endonuclease digestion analysis and DNA sequencing results showed that the 2 target segments were cloned into pSilencer4.1-CMV neo vector respectively. The results of RT-PCR indicated that both siRNA vectors could successfully knock down stathmin gene expression. CONCLUSION： The vector-based siRNA on stathmin gene can effectively knock down stathmin gene expression, which has the potential of being applied in gene therapy against malignant tumors.