人GLUT3基因RNA干扰病毒载体的构建与鉴定

Construction and identification of a lentiviral vector for RNA interference of human GLUT3 gene

目的：筛选出人GLUT3基因有效的RNA干扰（RNA interference,RNAi）序列,并构建出慢病毒RNAi载体。方法：根据GLUT3基因mRNA序列设计合成siRNA片段4个,分别定向克隆至pLV-shRNA载体上,并将构建的质粒转染HeLa细胞,运用实时荧光定量PCR（quantitative real-time PCR,qRT-PCR）检测GLUT3mRNA的表达量验证其干扰效果。筛选出其中有效的质粒与病毒包装质粒共转染293T细胞进行包装,收获并浓缩重组慢病毒颗粒,测定病毒颗粒滴度后,将病毒感染U251胶质瘤细胞以测定感染慢病毒干扰载体后胶质瘤细胞内GLUT3的表达情况。结果：重组RNAi质粒pLV-shRNA-GLUT3-1,pLV-shRNA-GLUT3-2,pLV-shRNA-GLUT3-3,pLV-shRNA-GLUT3-4经测序证实质粒载体构建成功;4个干扰质粒在HeLa细胞中均可以明显抑制GLUT3-mRNA的表达。pLV-shRNA-GLUT3可以在293T细胞中成功包装。收集293T细胞分泌的病毒上清浓缩后测定病毒颗粒LV-GLUT3滴度为1.5×10^9TU/mL。与感染阴性对照慢病毒颗粒（0.3641±0.044）相比,胶质瘤U251细胞感染慢病毒颗粒LV-GLUT3后,GLUT3蛋白相对表达明显降低（0.108±0.016,t=16.267,P〈0.001）。结论：成功构建了人GLUT3基因有效的慢病毒RNAi载体,为进一步研究GLUT3的生物学功能奠定了基础。

Objective：To construct an effective lentiviral vector for RNA interference（RNAi） with human glucose transporter 3（GLUT3） gene.Methods：Four pairs of shRNA sequences against different parts of GLUT3-mRNA were separately cloned into the RNAi plasmid vector pLV-shRNA by recombinant DNA technology to construct shRNA expression vectors pLV-shRNA-GLUT3-1,pLV-shRNA-GLUT3-2,pLV-shRNAGLUT3-3,and pLV-shRNA-GLUT3-4.The vectors were transfected into HeLa cells to detect the effectiveness of GLUT3 gene silencing.One of effective vectors was selected and co-transfected into 293 T cells with lentivirus packaging plasmids to obtain packaged lentivirus particles LVGLUT3.After viral titer determination,U251 glioblastoma cells were infected with LV-GLUT3 at a multiplicity of infection（MOI） of 10.Finally,the expression of GLUT3 protein was detected by Western blot.Results：DNA sequencing demonstrated that the shRNA sequences were successfully inserted into the pLV-shRNA vectors.In HeLa cells,the expression of GLUT3-mRNA was significantly downregulated by the recombinant vectors compared with negative control.The recombinant lentivirus LV-GLUT3 harvested from 293 T cells had a titer of 1.5×10^9 TU/mL.After infection with LVGLUT3,the expression of GLUT3 protein in U251 glioblastoma cells was down-regulated.Conclusion：An effective lentiviral shRNA expression vector targeting the GLUT3 gene is successfully constructed and can be used for further study on the functions of GLUT3 gene.