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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。
In this study, four target sequences were designed according to the DENN-SV sequence and shRNA design principles. After annealing, the target sequence was ligated with pRNAi-U6.1 / Neo empty vector by DNA recombination technology and transformed into competent E. coli. Amplify the strain, extract plasmid, digested, DNA sequencing identification. The recombinant eukaryotic expression vector was transfected into human breast cancer MCF-7 cells. The efficiency of DENN-SV mRNA expression was detected by RT-PCR and Western blot. The growth curve was drawn by MTT method. The sequencing result was the same as the designed sequence and was successfully transfected into human breast cancer MCF-7 cells. The expression of GFP (green fluorescent protein) was observed and both of them had inhibitory effect (P <0.01). The inhibitory effect of DS-1 group was the best The growth curve of MTT assay showed that the proliferation of cells in experimental group was significantly decreased (P <0.05). In this study, RNAi technology was successfully used to construct small interfering RNA recombinants, which laid the foundation for further study on gene therapy of breast cancer.