人HMGN5基因shRNA慢病毒载体构建与RNAi效率的鉴定

Construction of shRNA lentiviral vector targeting human HMGN5gene and identification of RNAi efficiency

目的构建人HMGN5基因的shRNA慢病毒载体并鉴定在肺癌A549和H1299细胞上的沉默效率。方法设计HMGN5基因特异性siRNA靶点,构建于慢病毒pLL-3.7载体,并筛选获得有效的shRNA慢病毒载体,在293T细胞包装成病毒颗粒,将其感染肺癌A549和H1299细胞,应用Real-time PCR方法从mRNA水平上检测HMGN5的沉默效率。结果构建的慢病毒载体shRNA的PCR鉴定和测序正确,包装病毒后滴度达到5×108TU/ml。shRNA慢病毒颗粒感染A549和H1299细胞后HMGN5基因的mRNA表达量较阴性对照载体慢病毒感染组分别下降了71.7%和50.7%。结论成功构建了HMGN5基因的shRNA慢病毒表达载体,在分子水平能够有效沉默靶基因,为探讨HMGN5在肿瘤基因治疗中的作用奠定了基础。

Objective To construct shRNA lentiviral vectors targeting humanHMGN5 gene and detect its effect of gene silence in A549 and H1299 cells.Methods The specific siRNA sequences targeting human HMGN5 gene were cloned into pLL-3.7 lentiviral vector.The lentivirus particles were packaged and HMGN5 specific shRNA was transmitted into A549 and H1299 cells after screeningfor the valid siRNA.HMGN5 efficiency of silencing was determined by Real-time PCR from mRNA level.Results It revealed shRNA plasmids was correctly constructed by PCR and sequencing.Virus was successfully packaged with a titer of 5×108TU/ml.HMGN5 expression could be down-regulated at mRNA level by virus infection in A549 and H1299 cells.It showed that HMGN5 mRNA decreased 71.7% in A549,and 50.7% in H1299.Conclusion The recombinant lentiviral shRNA has been successfully constructed,and it expressed vector targeting human HMGN5 gene.HMGN5 mRNA could be down-regulated effectively in A549 and H1299 cells,and it establishs the basis on further functional assay in cancer gene therapy.