靶向DENN-SV基因的shRNA真核表达载体的构建

Construction of shRNA Eukaryotic Expression Vector Targeting DENN-SV Gene

该研究根据DENN-SV序列及shRNA设计原则,设计四个靶点序列,退火后用DNA重组技术与pRNAi-U6.1/Neo空载体连接,转化到感受态E.coli中。扩增菌株,抽提质粒,进行酶切、DNA测序鉴定。将重组的真核表达载体转染人乳腺癌MCF-7细胞并使用RT-PCR及Western blot检测其抑制DENN-SV mRNA表达的效率,以MTT法绘制生长曲线。测序结果与设计序列相同,并已成功转染进入人乳腺癌MCF-7细胞,可见GFP(绿色荧光蛋白)表达,且都具有抑制作用(P<0.01),DS-1组的抑制效果最好;MTT法绘制生长曲线结果表明,实验组经该载体转染后细胞增殖程度显著减少(P<0.05)。该研究应用RNAi技术成功构建了小干扰RNA重组体,为进一步研究乳腺癌基因治疗奠定了基础。

This research designed four target sequences （DS-1, DS-2, DS-3, DS-4） based on DENN-SV sequence and shRNA design principles, and connected them to pRNAi-U6.1/Neo empty vector using recombinant DNA technology after annealing, and transformed them into competent E.coli. Amplify strains, extract plasmids and conduct digestion and DNA sequencing were obtained. After transfecting recombinant eukaryotic expression vector into human breast cancer MCF-7 cells, we used RT-PCR and Western blot inhibition to restrain the efficiency of DENN-SV mRNA expression, and got the growth curves of MCF-7 by MTT assay. Sequencing results conformed that the designed sequence had been successfully transfected into human breast cancer MCF-7 cells. The expression of GFP （green fluorescent protein） was visible but inhibited （P〈0.01）. The inhibitory effect of DS-1 group was the greatest. Results of growth curves by MTT assay showed that after transfection, the cell prolife ratio of experimen- tal group reduced significantly （P〈0.05）. The study successfully, with the application of RNAi technology, built up small interfering RNA recombinant, which laid the foundation for the further study of the breast cancer gene therapy.