慢病毒携带人Galectin-3基因RNAi的有效靶点筛选  

Construction and identification of RNA interference lentiviral expression vector of galectin-3 gene

作　　者：王明栋[1] 史彦芳[1] 王洪[1] 马文斌[2] 王任直[2] 

机构地区：[1]河北大学附属医院神经外科,河北保定071000 [2]北京协和医院神经外科,北京100730

出　　处：《南方医科大学学报》2008年第7期1264-1267,共4页Journal of Southern Medical University

基　　金：河北省科学技术研究与发展课题基金项目(052761123)

摘　　要：目的构建携带人Galectin-3基因的RNAi慢病毒载体,测定感染滴度,并对不同靶点进行有效筛选。方法根据Galectin-3基因设计4对,经退火制备的双链DNA oligo,连接经双酶切的线性化pGCL-GFP/U6载体,转化后经PCR鉴定,阳性克隆测序,质粒抽提,经CaCl2导入293T细胞,包装成慢病毒,收集病毒上清并检测其滴度。将含Galectin-3基因的RNAi病毒颗粒,感染靶细胞,荧光显微镜观察细胞荧光,感染率>50%时,收集细胞抽提RNA,RT-PCR检测Galectin-3mRNA水平,评价干扰效果。结果经测序、PCR分析证实目的序列成功连接到载体上,载体构建成功,包装成高滴度慢病毒。感染MCF-7细胞系后,细胞荧光显示感染率>50%,定量RT-PCR检测结果发现:1#和3#靶点敲减Galectin-3基因效率达95%、85%。结论成功构建并筛选了携带有人Galectin-3基因的RNAi慢病毒载体及有效靶点,为进一步研究Galectin-3在肿瘤细胞中的作用提供实验平台。Objective To Construct a recombinant lentiviral U6 plasmids for RNA interference （RNAi） of galectin-3 gene and select the optimal target sequence of galectin-3 gene for RNAi. Methods Double-stranded oligo DNAs were designed and synthesized according to the sequence of galectin-3 gene, and ligated into linearized pGCL-GFP/U6 plasmid followed by transformation into competent DH5α cells. After PCR and sequence analysis for verification of the positive clones, the plasmid pGCL-GFP/U6 Gal-3shRNA-1 was extracted and transfected into CaC12-treated 293T cells to obtain the viral vectors containing the RNAi sequence. MCF-7 cells were infected with pGCL-GFP/U6 Gal-3shDNA-1, and at the infection rate over 50%, the cells were harvested to extract the RNA, Real time-PCR was performed to determine the expression level of galectin-3 mRNA in the infected ceils. Results The recombinant vector was successfully constructed as confirmed by sequence analysis. High titer of the virus was obtained, and after infection of MCF-7 cells, RNAi targeting the 1#and 3# sequences in galectin-3 gene resulted in suppression of galectin-3 mRNA expression by 95% and 85%, respectively. Conclusion The recombinant lentiviral U6 plasmid for RNAi of Galectin-3 gene has been successfully constructed, which provides the basis for further study of the role ofgalectin-3 gene in tumor cells.

关 键 词：RNA干扰 半乳凝素-3基因 发夹结构 慢病毒 有效靶点筛选 

分 类 号：R73-36[医药卫生—肿瘤]