慢病毒载体介导的层黏连蛋白受体稳定抑制细胞株的建立

Construction of Laminin Receptor Inhibited Stable Cell Line Mediated by Lentivirus Vector

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 ＆amp; 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.