摘要
本研究旨在构建kir2ds4基因RNAi慢病毒载体。针对已经筛选确定的kir2ds4基因RNAi有效靶序列,合成靶序列的OligoDNA,退火形成双链DNA,与经HpaI和XhoI酶切后的pSicoR-GFP载体[含U6启动子和绿色荧光蛋白(GFP)]连接产生LV-shkir2ds4慢病毒载体,应用PCR筛选阳性克隆并进行测序鉴定。用LV-shkir2ds4,包装系统质粒共转染包装细胞293T细胞,产生慢病毒颗粒,以293T细胞GFP蛋白的表达水平测定病毒滴度。结果表明,PCR和测序结果证实成功地构建了kir2ds4shRNA的慢病毒载体LV-shkir2ds4。293T细胞测定病毒滴度为6×108TU/ml。结论:成功地构建了人kir2ds4基因RNAi慢病毒载体。
This study was aimed to construct a lentiviral vector of RNA interfered （RNAi）-kir2ds4 gene. In accordance with study-confirmed effective sequence of siRNA targeting kir2ds4 gene, the complementary DNA containing both sense and antisense oligonuctide of the targeting sequence was designed, synthesized and inserted into pSicoR-GFP vector containing U6 promoter and GFP sequence. The resulting lentiviral vector containing kii＇2ds4 shRNA was named as LV-sh kir2ds4, and confirmed by PCR and sequencing. 293T cells were co-transfected with lentiviral vector LV-sh kir2ds4 and packaging system. All virus stocks were produced by Lipofectamine 2000 -mediated transfection. The titer of virus was tested according to the expression level of GFP. As a result, PCR and DNA sequencing demonstrated that the lentivirus RNAl vector of kir2ds4 was constructed successfully. The titer of virus tested by expression level of GFP was 6 × 10^8 TU/ml . It is concluded that the lentivirus RNAi vector of kir2ds4 has been successfully constructed.
出处
《中国实验血液学杂志》
CAS
CSCD
2008年第3期663-666,共4页
Journal of Experimental Hematology
基金
全军医学科研重大项目
编号01MB068
