靶向CCR7基因shRNA表达载体的构建及鉴定  

Construction and identification of pGC-silencer-CRM30 CCR7 short hairpin RNA expression vectors.

作　　者：孙仁虎[1] 李疆[1] 崔静[1] 吕清[1] 刘兴华[1] 王国斌[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院腹腔镜中心,武汉430022

出　　处：《华中医学杂志》2009年第1期6-8,27,共4页Central China Medical Journal

基　　金：国家自然科学基金资助项目(No.30772128)

摘　　要：目的设计并构建靶向CCR7基因shRNA真核表达质粒,并检测其干扰效果。方法以CCR7为靶基因设计具有短发夹结构的模板寡核苷酸,退火形成互补双链结构,再克隆至pGC-silencer-CRM30载体,构建的3条重组短发夹shR-NA表达载体分别命名为pGC-silencer-CRM30-CCR7A、-CCR7B和-CCR7C。采用测序法鉴定寡核苷酸序列。将构建好的真核表达载体转染人结肠癌SW480细胞,荧光显微镜观察转染效果,RT-PCR法检测CCR7干扰效率。结果重组质粒测序结果与Genebank中的CCR7cDNA序列相符,转染SW480细胞后,荧光显微镜下可观察到绿色荧光蛋白,RT-PCR结果表明,pGC-silencer-CRM30-CCR7C干扰效果最强。结论靶向CCR7基因shRNA表达载体构建无误,为进一步探讨趋化因子受体CCR7在胃肠道恶性肿瘤生物学行为中的作用奠定了基础。Objective To construct CCR7 shRNA eukaryotic expression vectors to transfect into SW480 cells in order to further study the silencing effects of the vector on the targeting gene CCR7. Methods The shRNA oligonucleotides targeting for CCR7 gene were synthesized and cloned into pGC-silencer-CRM30 to generate shRNA eukaryotic expression vectors. The recombinant named as pGC-sileneer-CRM30-CCR7 A, -CCR7 B and -CCR7 C. ShRNA expression plasmids were identificated by sequencing. The eukaryotic expression vectors were transfected into SW480 cells. The green fluorescent protein （GFP） was detected by fluorescence microscope, and the silencing effects of the recombinant vectors were determined by RT-PCR. Results The recombinant sequence identified by sequencing was the same as the targeting one. In the SW480 cells transfected with the recombinant vectors, the expression of GFP was detected, the silencing effects of pGC-silencer-CRM30-CCR7 C was more evident than other recombinant vectors. Conclusion shRNA recombinant was established successfully by RNAi technique and transfected into SW480 cells.

关 键 词：RNA干扰 短发卡RNA 趋化因子 CCR7 

分 类 号：R73-3[医药卫生—肿瘤]