S100P基因shRNA慢病毒载体构建与RNA干扰效率鉴定

Construction of shRNA lentiviral vector targeting S100P gene and identification of its siRNA efficiency

目的构建S100P基因短发夹RNA(shRNA)慢病毒载体,并在胃癌细胞中鉴定其沉默效率。方法设计S100P基因特异性的RNA干扰序列,构建shRNA慢病毒载体,与S100P过表达质粒共转染293T细胞。随后筛选有效的shRNA慢病毒载体,与pHelper 1.0和pHelper 2.0质粒共转染293T细胞,包装病毒,感染胃癌细胞株MGC-803和SGC-7901。采用RT-PCR和Western blot检测S100P基因的敲减效率。结果成功构建S100P基因的shRNA慢病毒载体,MGC-803和SGC-7901细胞中S100P基因的mRNA和蛋白表达均降低(P<0.05)。结论成功构建的S100P基因shRNA慢病毒载体能够在细胞水平有效沉默靶基因。

Objective To construct a short hairpin RNA（sh_RNA） lentiviral vector targeting human SLOOP gene and identify its silence effect in gastric cancer cells. Methods Five specific siRNA sequences targeting human SLOOP gene were designed and cloned into GVllS-GFP lentiviral vectors, which were co-transfected with overexpressing vector of S100P into 293T cells. The valid shRNA lentiviral vectors were screened and transfected with pHelper 1.0 and pHelper 2.0 plasmids into 293T cells for virus packaging. Then gastric cancer cell lines MGC-803 and SGC-7901 were infected with specific shRNA lentivirus, and the mRNA and protein expressions of S100P gene were detected by RT-PCR and Western blot, respectively. Results shRNA lentiviral vector targeting human SLOOP gene was successfully constructed, and the mRNA and protein expressions of S100P gene were significantly decreased in MGC-803 and SGC-7901 cells （P〈0. 05）. Conclusion The recombinant shRNA lentiviral vector targeting SLOOP gene can effectively silence target gene at the cellular level.