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目的:构建针对人survivin基因的siRNA真核表达载体,检测其对人乳腺癌SKBr-3细胞中survivin基因表达的干 涉作用。方法: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,把重组质粒稳定转染入SKBr-3细胞,RT-PCR、Western blot印记杂交和细胞免 疫化学法检测其对mRNA和蛋白表达的干涉效果。结果:经酶切鉴定及基因测序证实,重组质粒中已插入目的基因片段。 RT-PCR显示两个干涉载体均抑制了目的基因的转录;Western blot印记杂交和细胞免疫化学检测结果显示pSUPER-S1对蛋 白表达的抑制作用更强。结论:成功地构建了针对人survivin基因的RNA干涉真核表达载体pSUPER-S1和pSUPER-S2,并在 人乳腺癌细胞株SKBR-3中有效发挥了对survivin基因的干涉作用。
OBJECTIVE: To construct siRNA eukaryotic expression vector targeting human survivin gene and investigate its interference effect on survivin gene expression in human breast cancer SKBr-3 cells. Methods: The synthesized oligonucleotide strands were annealed to double strands and ligated into pSUPER eukaryotic expression vector which was digested with Hind Ⅲ and Bgl Ⅱ. The recombinant plasmid was digested and sequenced. The recombinant plasmids were stably transfected into SKBr-3 cells by lipofectamine. The interference effect on mRNA and protein expression was detected by RT-PCR, Western blot and immunocytochemistry. Results: The results of restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid had inserted the target gene fragment. RT-PCR showed that both interfering vectors inhibited the transcription of the target gene; Western blotting and immunocytochemistry showed that pSUPER-S1 inhibited the protein expression more. CONCLUSION: The RNA interference eukaryotic expression vectors pSUPER-S1 and pSUPER-S2 targeting human survivin gene were successfully constructed and the interference of survivin gene was effectively exerted in human breast cancer cell line SKBR-3.