GPC3基因重组慢病毒载体的构建及其在肝癌细胞中的表达  

Construction of shRNA Lentiviral Vector Targeting Human GPC3 Gene and Its Expression in HepG2 Cells

作　　者：祁馨卉[1] 崔慧霞[1] 马楠[1] 姜又红[1] 

机构地区：[1]中国医科大学附属第一医院肿瘤研究所二室,沈阳110001

出　　处：《中国医科大学学报》2013年第8期726-729,共4页Journal of China Medical University

基　　金：辽宁省科学技术计划项目(2011415052-3)

摘　　要：目的构建含有GPC3的shRNA序列慢病毒表达载体并转染肝癌细胞,观察其转染效率及在肝癌细胞中的表达。方法应用重组DNA技术,将设计好的GPC3基因特异性siRNA序列构建至慢病毒表达载体pGLV3-GFP上,并应用脂质体法转染293T细胞,进行病毒包装及滴度测定。采用RT-PCR及Western blot分别从mRNA和蛋白水平检测转染HepG2细胞后GPC3基因的表达情况。结果慢病毒载体构建成功,并测定滴度为1×108TU/mL,转染后,GPC3基因表达明显降低。结论成功构建GPC3基因的shRNA慢病毒载体,所介导的RNAi能有效抑制靶细胞中GPC3基因的表达。Objective To construct shRNA lentiviral vectors targeting human GPC3 gene and observe the transfection and expression level in HepG2 cells.Methods The siRNA sequences specifically targeting the GPC3 gene were designed and cloned into lentiviral vector pGLV3-GFP using DNA recombinant technique.The 293T cells were transfected by lipofectin reagent for lentiviral particles packaged,and viral titer was determined.GPC3 mRNA and protein expression were examined by RT-PCR and Western blot.Results The recombinant lentiviral vectors were successfully constructed and the virus reached a titer of 1×108 TU/mL.The expression of GPC3 was significantly decreased after infection with the lentivirus.Conclusion shRNA expressing lentiviral recombinants targeting the GPC3 gene were successfully constructed,which provides a basis for the further functional study of GPC3.

关 键 词：GPC3 RNA干扰 慢病毒载体 

分 类 号：R73[医药卫生—肿瘤]