人Smurf1 shRNA真核表达载体的构建与鉴定

目的：构建三条人Smurf1 shRNA真核表达载体,以逆转TGF-β1诱导人HK-2细胞转分化。方法：针对人Smurf1基因设计,采用体外转录生物合成的特异性短发夹RNA（shRNA）双链分子,对重组质粒（命名为pSilencer-S1 shRNA、pSilencer-S2 shRNA、pSilencer-S3 shRNA）进行1%琼脂糖凝胶电泳,初步鉴定及测序鉴定。结果：1%琼脂糖凝胶电泳初步鉴定证实Smurf1 siRNA表达载体克隆构建成功,插入序列测序结果与合成序列结果一致。结论：Smurf1 pSilencer-S1 shRNA、pSilencer-S2 shRNA、pSilencer-S3 shRNA真核表达载体构建成功。

Construction and Identification of Human Smurf1 shRNA Eukaryotic Expression Vectors

Objective： To construct three recombinant plasmids of Smurf1 shRNA eukaryotic expression vectors for retroconversion of TGF-β1-stimulated transdifferentiation of human HK-2 cells.Methods： Short hairpin RNAs（shRNAs） which specificially targeted for three sequences（TACGTCCGGTTGTATGTAA,TGAAGGAACGGTGTATGAA,and GCAAGGCTAGATTCGAAGT） of Smurf1,were designed and synthesized.The recombinants（named pSilencer-S1,pSilencer-S2,and pSilencer-S3） were finally sequenced and identified by restriction endonuclease digestion.Results： Smurf1 siRNA expression vectors were successfully constructed and identified by double endonuclease digestion.Sequence analysis showed that the inserted fragments were coincident with the sequences of synthesized siRNA oligonucleotides.Conclusions： Smurf1 siRNA expression vectors have been successfully constructed.