摘要
目的构建特异性沉默SMO基因的重组慢病毒载体,筛选对T293细胞SMO基因表达抑制效率最高的siRNA。方法根据SMO基因信息,设计了4个小干扰序列和1个阴性对照序列,利用慢病毒质粒载体pGCSIL-GFP构建了5个重组质粒。用脂质体将重组慢病毒载体转染293T细胞后,Western blot检测沉默效率,从中筛选沉默效率高的质粒载体进行慢病毒颗粒大量包装。结果测序结果证明4个重组慢病毒质粒载体pGCSIL-GFP-721、pGCSIL-GFP-722、pGCSIL-GFP-723、pGCSIL-GFP-724的插入序列完全正确,重组慢病毒载体转染293T细胞后,Western blotting证实重组慢病毒质粒载体pGCSIL-GFP-723的干扰效率最高。包装获得Lv-SIL-SMO723慢病毒颗粒。结论成功筛选获得特异性抑制SMO的siRNA,成功构建了特异性沉默SMO基因慢病毒载体,其产生的慢病毒颗粒能高效特异地沉默SMO基因,为进一步应用奠定基础。
Objective To screen and construct a recombinant lentivirus vector of RNA interference targeting SMO gene（NM_005631） in order to select the most efficient siRNA to express SMO in T293 cells.Methods According to Genbank information of SMO,four RNA interfering sequences and a negative sequence were designed and inserted into plasmid pGCSIL-GFP.Recombinant lentivirus plasmid was transfected into 293T cell line by liposome.Western blot was then used to investigate the interfering efficiency.The recombinant lentivirus plasmid with high interfering efficiency was cotransfected with packing vector in 293T cell line.Results Sequence result showed that recombinant lentivirus plasmids pGCSIL-GFP-721,pGCSIL-GFP-722,pGCSIL-GFP-723,pGCSIL-GFP-724 were constructed successfully.After the transfaction with liposome,Western blotting analysis confirmed that pGCSIL-GFP-723 had the highest interfering efficiency.The high interfering efficiency plasmid was named by Lv-SIL-SMO723 after packing.Conclusion Recombinant lentivirus plasmid of RNA interference targeting SMO gene was successfully constructed,which could suppress SMO expression with high efficiency,and can be used in further study.
出处
《同济大学学报（医学版）》
CAS
2009年第2期15-18,共4页
Journal of Tongji University(Medical Science)
基金
国家自然科学基金资助项目(30500506)
