雌激素受体α基因shRNAi慢病毒载体的构建与滴度测定

Construction of Lentiviral Vector for ERα shRNAi and Titer Identification of Virus

目的:构建雌激素受体(ER)α基因RNA干扰(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EcoR Ⅰ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。结果: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2×108TU/mL。结论: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基础。

Objective： To construct a lentiviral vector of RNA interference （RNAi） of estrogen receptor （ER）α gene and detect the fiter of virus-infected cells. 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, which was confirmed by PCR and sequence analysis. HEK-293T ceils 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： Results： PCR and DNA sequencing demonstrated that shERα-LV producing ERα-shRNA was constructed successfully. The titer of concentrated virus was 2×10^8 TU/mL. Conclusion： The lentivirus RNAi vector of ERα-was constructed successfully, which provided the basis for ERα signal transduction pathway in breast cancer.