人CCL5基因RNA干扰慢病毒载体的构建

目的 构建人CCL5基因RNA干扰(RNAi)慢病毒载体.方法 根据人CCL5基因信息,构建4个携带RNAi序列的pGCSIL-GFP质粒,与pHelper 1.0、Helper 2.0质粒一起利用293T细胞进行慢病毒包装.用CCL5 RNAi慢病毒载体感染人宫颈癌细胞(Hela),使用RT-PCR方法验证其干扰效率.结果 4个靶点中有3个靶点(a1、a2、a3)在Hela细胞上对CCL5基因的表达都有非常显著的敲减效果,敲减效率均达到95%以上.结论 构建的CCL5 RNAi慢病毒载体在Hela细胞中有较高的敲减效率,提示RNAi技术能够使细胞CCL5基因沉默.

Construction and identification of lentiviral vector of the RNA interference of human CCL5 gene

Objective To construct a lentiviral vector of RNA interference of chemokine CC motif ligand-5 (CCLS) gene and identify its knockout effectiveness in Hela cells. Methods The pGCSIL-GFP plasmids which expressed four RNAi sequences ( a1, a2, a3, a4) respectively for human CCL5 gene togeth-er with pHelper 1.0 and Helper 2.0 plasmids were transfected into 293T cells in order to construct tentivi-ral vectors of RNA interference of CCL5 gene. Then the lentiviral vectors were transfected into Hela cells, and RT-PCR was used to evaluate its knockout effectiveness for CCL5. Results The knockout effective-ness of a1 ,a2 or a3 was beyond 95% excepting a4 ,whose knockout effectiveness was beyond 90% in Hela cells. Conclusion The knockout effectiveness of the constructed lentviral vectors for human CCL5 was satisfactory. Our results demonstrated that RNAi is able to silence the potential key gene CCL5 in human cancer cells.