膜-细胞骨架联接分子ezrin shRNA 真核表达载体的构建
Construction of Eukaryotic Expression Vector of Short Hairpin RNA for ezrin
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摘要: 目的 构建膜-细胞骨架联接分子(ezrin)特异的RNA干扰质粒载体。 方法 设计转录短发夹状RNA (short hairpin RNAs, shRNA)的DNA 序列，与pSUPER质粒载体连接；将构建成功的特异性表达载体(pSUPER-ezrin)转染至人乳腺癌MCF-7细胞系，并应用RT-PCR和Western blot检测ezrin的表达。 结果 与MCF-7细胞和转染空白质粒pSUPER细胞相比，转染pSUPER-ezrin表达载体的MCF-7细胞ezrin mRNA和蛋白的表达明显降低。 结论 构建的ezrin特异性shRNA表达载体可有效地沉默ezrin基因，为进一步研究ezrin表达对乳腺癌转移的影响奠定了基础。Abstract: Objective To construct the short hairpin RNA （shRNA） expression vector specific for ezrin. Methods Oligonucleotides were designed specific for ezrin gene. After annealing, the formed double-stranded DNAs were ligated with pSUPER. The expression vectors of pSUPER -shRNA were identified by enzyme digestion and sequence analysis and transfected into MCF-7 cells. The expression of ezrin was analyzed by RT-PCR and Western blot. Results The vector was identified by restriction enzyme digestion and sequence analysis. The expression vectors of pSUPER-shRNA was successfully constructed and transfected into MCF-7 cells. The ezrin expression was significantly suppressed after transfection with ezrin shRNA vector. Conclusion The pSUPER -shRNA specific for ezrin is successfully constructed and it will be helpful for further study on the significance of ezrin on the metastasis of breast cancer.
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