Slug基因RNAi慢病毒载体的构建与鉴定

Construction and identification of a lentiviral vector mediating RNA interference of Slug gene

目的构建Slug基因RNA干扰(RNA interference,RNAi)慢病毒表达载体。方法针对人Slug基因的序列,设计出RNA干扰的靶序列,合成靶序列Oligo DNA,退火形成双链DNA,通过Age I和EcoR I酶切后的pGCSIL-GFP载体连接产生shRNA慢病毒载体,质粒转化感受态细菌,筛选阳性克隆并用插入鉴定引物进行PCR鉴定阳性克隆并测序,同时应用Real-time PCR和Western blot方法检测HCT116结肠癌细胞中Slug基因mRNA和蛋白的表达情况。结果 PCR鉴定与DNA测序证实合成的含Slug shRNA慢病毒载体寡核苷酸链插入正确。Western blot证实Slug RNAi慢病毒载体能够抑制Slug的表达。结论成功构建Slug基因RNAi慢病毒表达载体,为后续感染结肠癌细胞,为探索在结直肠癌发生和发展中的作用奠定基础。

For further study on the function of Slug gene,we constructed a lentiviral expression vector of Slug RNA interference（RNAi） of human.Towards the Slug gene sequences,RNAi target sequences were designed,then the target sequences of Oligo DNA were synthesized and annealed to double stranded DNA,which was subsequently connected with Age I/EcoR I-digested pGCSIL-GFP vector to constructed lentiviral vector pGCSIL-GFP-shSlug.The vector was transformed into competent E.coli cells,and the candidate clones were identified by PCR and sequencing.Western blotting was used to detect the expression of Slug protein in the transfected cells,and Real-time PCR was employed to detect Slug mRNA expression.Western blotting proved the presence of Slug expression in the transfected HCT116 cells.We conclude that the lentiviral vector for Slug has been successfully constructed with a high yield of lentivirus,which facilitate further investigation of the roles of Slug gene in the development and progression of colorectal cancer.