靶向细胞周期蛋白E的shRNA真核表达载体的构建和鉴定_医学论文.docVIP

靶向细胞周期蛋白E的shRNA真核表达载体的构建和鉴定_医学论文 靶向细胞周期蛋白E的shRNA真核表达载体的构建和鉴定_医学论文 【摘要】 　　［目的］ 构建靶向细胞周期蛋白E的shRNA真核表达载体。［方法］ 针对cyclin E基因，设计1对寡核苷酸，形成双链后将其将其插入Psilencer2.1-U6-neo质粒，构建cyclin E shRNA真核表达载体，酶切和测序鉴定。转染MCF-7细胞株，应用western blot检测cyclin E蛋白的表达。［结果］ 酶切和测序结果显示，成功构建特异性针对cyclin E的shRNA 表达质粒。转染MCF-7细胞后下调cyclin E的表达。［结论］ 成功构建靶向cyclin E的shRNA真核表达载体，为进一步应用RNAi技术进行肿瘤基因治疗的研究提供实验基础。 【关键词】 细胞周期蛋白E　短发卡结构状RNA　RNA干扰 　　 Construction and Identification of shRNA Expression Vector Specific for Cyclin E Abstract: ［Purpose］ To construct the short hairpin RNA （shRNA） expression vector specific for cyclin E. ［Methods］ Oligonucleotides were designed specific for cyclin E gene, after annealing the formed double-stranded DNAs were ligated with Psilencer2.1-U6-neo. The expression vectors of Psilencer2.1-U6/shRNA were identified by enzyme digestion and sequence analysis and transfected into MCF-7 cells. The expression of cyclin E was analyzed by Western blot. ［Results］ The vector was identified by restriction enzyme digestion and sequence analysis, the expression vectors of Psilencer2.1-U6/shRNA was successfully constructed. The expression of MCF-7 cells cyclin E expression level was suppressed after transfection with cyclin E shRNA vector. ［Conclusion］ The Psilencer2.1-U6/shRNA specific for cyclin E is successfully constructed and it will provide experimental basis for cancer gene therapy by RNAi technique. Key words: cyclin E short hairpin RNA RNA interference 肿瘤是一类细胞周期性疾病，细胞生长周期的失调在肿瘤的发生发展中起关键作用。Cyclin E高表达可以缩短G1期，引起中心体过增殖，扰乱有丝分裂，形成不稳定的染色体，从而诱发肿瘤[1]。已有研究提示，cyclin E在白血病、乳腺癌、结肠癌、淋巴瘤等多种肿瘤中过表达[2]，因此，cyclin E有望成为肿瘤基因治疗的新靶点。RNA干扰(RNA interference，RNAi)技术是一种能阻断目的基因表达的有效方法[3]。本研究设计了靶向cyclin E的短发卡结构状RNA (short hairpin RNA，shRNA)，利用载体质粒Psilencer2.1-U6-neo构建产生shRNA，为今后将其进一步应用于cyclin E的靶向治疗研究奠定了基础。 1 材料与方法 1.1 材 料 DMEM培养基、胰蛋白酶、E.coli DH5α、脂质体转染试剂LIPOFECTAMINE 2000 REAGENT (Invitrogen)、胎牛血清(杭州四季青生物材料有限公司)，鼠抗人cyclin E多克隆抗体、HRP标记的兔抗鼠IgG为Santa cruz公司产品、Supersignal West pico Trial Kit为PIERCE公司产品，小量质粒提取试剂盒（中鼎公司