敲低CTCF腺病毒表达载体的构建及效果检测  

Construction of an Adenovirus Vector for CTCT Small-Interfering RNA and its Knockdown Effect Detection

作　　者：王天艺[1] 张彦[1] 师明磊[1] 赵志虎[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2014年第4期484-487,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31370762)

摘　　要：目的:获得敲低效果较好的CCCTC结合因子(CTCF)的RNA干扰腺病毒载体,以便于研究其在肿瘤发生发展中的作用。方法:从已发表文献中获得CTCF敲低靶序列,合成2对含有小发卡结构的寡核苷酸序列,将其进行退火磷酸化后,分别克隆到腺病毒包装载体上;将重组质粒转染人胚肾293A细胞,收获腺病毒;将收获的腺病毒分别感染人胚肾HEK293细胞和靶细胞人肺腺癌细胞A549,通过RT-PCR和Western印迹鉴定相关基因的表达变化。结果与结论:RT-PCR和Western印迹鉴定显示构建的表达CTCF短发夹RNA(shRNA)的腺病毒载体能够有效抑制CTCF转录和蛋白水平,为后续CTCF的生物学功能和机制研究奠定了基础。Objective： To obtain high effective small interfering RNA（siRNA） interference vector of CCCTC bind-ing factor（CTCF） gene for studying the biological function of CTCF in the development and progression of tumor. Methods： CTCF siRNA was designed and constructed based on published papers and the synthesized sequences were annealed to form double-strand oligonucleotide and cloned into interference vector. Then the interference vec-tors were transfected into 293A cells. The knockdown effect was detected by RT-PCR and Western blotting. Re-sults ＆ Conclusion： The adenoviruses containing short hairpin RNA（shRNA） targeting the CTCF gene have been successfully constructed, which laid the foundation for further study of CTCF function.

关 键 词：CCCTC结合因子 RNA干扰 腺病毒载体 

分 类 号：Q78[生物学—分子生物学]