siRNA表达载体对人HeLa细胞中caspase-8基因的干涉作用

The interference effect of vector-based siRNA on caspase-8 gene

目的:构建针对人caspase-8基因mRNA的siRNA表达载体,并观察其对转染细胞中的caspase-8的抑制作用。方法:化学合成用于产生针对caspase-8的发夹状RNA的寡核苷酸,将合成的寡核苷酸链退火形成双链,连接入经HindⅢ和BglⅡ双酶切后的pSUPER真核表达载体。对重组质粒进行酶切分析和测序鉴定。通过脂质体介导,把重组质粒瞬时转染入HeLa细胞,RT-PCR、Western blot以及immunofluorescence staining检测其对mRNA和蛋白表达的干涉效果。结果:成功地构建了针对人caspase-8基因的RNA干涉真核表达载体pSUPER-C1和pSUPER-C2,并在人HeLa细胞中有效发挥了对caspase-8基因的干涉作用,而且pSUPER-C1对于caspase-8基因的抑制作用要优于pSUPER-C2。结论:成功地构建了针对人caspase-8基因的siRNA载体,转染HeLa细胞后可抑制caspase-8基因的表达。

Objective： To construct the eukaryotic expression vector for RNA interferencing human caspase -8 gene and detect its interference effect HeLa cell line. Methods ：Two target gene segments were synthesized and cloned into pSUPER vector respectively to construct two recombinant eukaryotic expression vectors：pSUPER -C1 and pSUPER - C2. The two recombinant vectors were identified by enzyme digestion analysis and DNA sequencing. Then He- La cells were transfected with pSUPER - C1 or pSUPER - C2, the interference effect was detected by RT - PCR , Westernblot and immunofluorescence staining. Results： The results of RT - PCR, Western blot and immunofluorescence staining indicated that both vectors could knock down the transcription and expression of caspase gene, and that pSUPER- C1 had better interference effect than pSUPER -CS2. Conclusion：The transcription and expression of caspase- 8 gene were inhibited effectively by the constructed RNAi eukaryotic expression vectors in the HeLa cells.