摘要
目的:构建靶向层黏连蛋白受体(LR)基因的小发卡RNA(sh RNA)慢病毒表达载体,鉴定其对LR的抑制效果,并筛选LR稳定抑制的He La细胞株。方法:设计针对LR的sh RNA序列,将此序列和H1启动子克隆入含有EGFP报告基因的p Lenti6/v5慢病毒表达载体,通过病毒包装、细胞感染、抗生素筛选获得稳定细胞株,用real-time PCR和Western印迹检测筛选得到的稳定细胞株中LR的表达水平。结果和结论:构建了含有LR靶向sh RNA的慢病毒表达载体,包装成病毒后感染He La细胞,经抗生素筛选后获得了稳定抑制LR的细胞株;筛选后的细胞均可观察到报告基因EGFP的表达;经m RNA和蛋白水平检测,LR-sh6和LR-sh7均可显著抑制He La细胞株中LR的表达。
Objective: To construct the lentiviral vector expressing laminin receptor(LR) targeted small hairpin RNA(shRNA) and the stable LR inhibited HeLa cells. Methods: shRNA sequences were designed, and together with H1 promoter were cloned into pLenti6/v5 vector with EGFP report gene. After virus packaging, infecting and cell screening, real-time PCR and Western blot analysis were conducted to determine LR expression in HeLa cells. Results & Conclusion: The lentiviral vectors carrying LR shRNA were successfully constructed. LR knocked-down stable HeLa cell lines were established and positive clones were observed to express EGFP. Real-time PCR and Western blot analysis indicated that both LR-sh6 and LR-sh7 could suppress LR expression effi?ciently.
出处
《生物技术通讯》
CAS
2014年第6期796-799,共4页
Letters in Biotechnology