细胞周期蛋白E siRNA真核表达载体的构建与鉴定

目的构建细胞周期蛋白E(Cyclin E)小干扰RNA(siRNA)的真核表达载体,并检测其抑制乳腺癌细胞株MCF-7中Cyclin E表达的效果。方法根据基因文库中Cyclin E的cDNA序列,设计并合成两段siRNA,分别转入到pGENESIL-1质粒中,并对重组质粒(命名为pGENESIL/Cyclin E-1和pGENESIL/Cyclin E-2)进行测序鉴定。脂质体介导下转染MCF-7细胞株,RT-PCR检测转染后Cyclin E的表达。结果Cyclin E siRNA真核表达载体构建成功,测序结果显示插入片段的方向和序列与预想一致。RT-PCR检测显示Cyclin E的表达显著降低。结论Cyclin E siRNA真核表达载体的成功构建为进一步研究以Cyclin E为靶位点对乳腺癌进行基因治疗奠定了重要的实验基础。

Construction and Identification of Cyclin E siRNA Eukaryotic Expression Vectors

Objective To construct eukaryotic expression vectors of siRNA tageting Cyclin E, and measure its inhibition efficiency in cyclin E expression of the human breast cancer cell line MCF-7. Methods Two pairs of siRNA were designed and synthesized according to the cyclin E cDNA sequence in Genebank, and they were then inserted into pGENESIL-1 plasmids respectively. The recombinants (named pGENESIL/Cyclin E-1 and pGENESIL /Cyclin E-1) were sequenced and identified. The vector was transfected into MCF-7 cell line, and RT-PCR was performed to examine the expression of cyclin E. Results Cyclin E siRNA eukaryotic expression vectors were successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragments revealed that the sequences were identical to those of synthesized siRNA oligonucleotides. Conclusion Construction of cyclin E siRNA eukaryotic expression vectors lays an experimental foundation for gene therapy of breast cancer by tageting Cyclin E.