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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 a shRNA lentiviral vector encoding human HMGN5 gene and identify its silencing efficiency in lung cancer A549 and H1299 cells. Methods The specific siRNA targeting HMGN5 gene was designed and constructed on the lentiviral vector pLL-3.7. The shRNA lentiviral vector was screened and transfected into 293T cells and infected with A549 and H1299 cells. Real-time PCR method to detect the silencing efficiency of HMGN5 from the mRNA level. Results The constructed lentiviral vector shRNA was identified by PCR and sequenced correctly. After virus was packaged, the titer reached 5 × 108TU / ml. The mRNA expression of HMGN5 gene in A549 and H1299 cells transfected with shRNA lentivirus particles decreased 71.7% and 50.7% compared with the negative control vector lentivirus group respectively. Conclusion The shRNA lentiviral expression vector of HMGN5 gene was successfully constructed and the target gene could be effectively silenced at the molecular level, which laid the foundation for exploring the role of HMGN5 in gene therapy of cancer.