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ERα基因shRNAi慢病毒载体的构建与滴度测定

曾宪旭,班振英,党秋红,楚天骄,雷冬梅   

  1. 郑州大学第三附属医院病理科
  • 收稿日期:2011-12-07 修回日期:2012-06-15 出版日期:2012-11-15 发布日期:2012-11-15
  • 通讯作者: 曾宪旭

Construction and identification of lentiviral vector of RNA interference of ER? gene

  • Received:2011-12-07 Revised:2012-06-15 Published:2012-11-15 Online:2012-11-15

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摘要： 目的：构建ERα基因RNAi慢病毒载体,检测病毒感染细胞的滴度。方法：针对已经筛选确定的ERα基因RNAi有效靶序列，合成靶序列的Oligo DNA，退火形成双链DNA，与经Age I和EcoRI 酶切后的pGCsil-GFP载体［含U6启动子和绿色荧光蛋白（GFP）］连接产生shERα-LV慢病毒载体，PCR筛选阳性克隆，测序鉴定。 用shERα-LV载体、pHelper 1.0 载体和pHelper 2.0质粒共转染包装细胞293T细胞，包装产生慢病毒，以293T细胞GFP蛋白的表达水平测定病毒滴度。结论：PCR和测序证实，成功构建ERα shRNA的慢病毒载体shERα-LV. 包装慢病毒，浓缩病毒悬液的滴度为2E+8TU/ml。讨论：成功构建人ERα基因RNAi慢病毒载体，为ERα在乳腺癌发生发展中的研究提供了工作基础。

关键词: RNA干扰, ERα, 质粒构建, 慢病毒

Abstract: AIM: To construct a lentiviral vector of RNA interference (RNAi) of ERα gene and transfect them into HEK-293T cells to detect the titer of virus. METHODS: The effective sequence of siRNA targeting ERα gene was confirmed in our previous study. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing ERα?shRNA was named shERα-LV, and it was confirmed by PCR and sequencing. HEK-293T cells were cotransfected with lentiviral vector shERα-LV，pHelper 1.0 and pHelper 2.0. All virus stocks were produced by LipofectamineTM 2000 transfection. The titer of virus was tested according to the expression level of GFP. CONCLUSION: PCR and DNA sequencing demonstrated that shERα-LV producing ERα-shRNA was constructed successfully. The titer of concentrated virus was 2E+8TU/ml. DISCUSSION: The lentivirus RNAi vector of ERα?was constructed successfully. Our work provides the basis for ERα signal transduction pathway in breast cancer.

Key words: RNA interference, ERα, Plasmid construction, Lentivirus