论文部分内容阅读
目的构建CDC25B3基因RNAi慢病毒载体。方法合成CDC25B3基因RNAi有效靶序列的OligoDNA,退火形成双链DNA,与双酶切后的pGCL-GFP载体(含有U6启动子和GFP基因)连接产生LV-shCDC25B3慢病毒载体,挑选阳性克隆经PCR和测序鉴定。用脂质体转染法将LV-shCDC25B3、pHelper1.0和pHelper2.0质粒共转染293T细胞,产生慢病毒,以293T细胞绿色荧光蛋白的表达水平测定病毒滴度。结果PCR和测序证实,成功地构建了CDC25B3shRNA的慢病毒载体LV-shCDC25B3。浓缩病毒悬液的滴度为1×108TU/ml。结论成功构建人CDC25B3基因RNAi慢病毒载体。
Objective To construct RNAi lentiviral vector of CDC25B3 gene. Methods Oligo DNA of RNAi effective target sequence of CDC25B3 gene was synthesized and annealed to form double-stranded DNA. The double-stranded DNA was ligated with double-digested pGCL-GFP vector (containing U6 promoter and GFP gene) to generate LV-shCDC25B3 lentiviral vector. And sequencing identification. The lentivirus was co-transfected with 293T cells by liposome transfection method with LV-shCDC25B3, pHelper1.0 and pHelper2.0 plasmids. The virus titer was determined by the expression level of green fluorescent protein of 293T cells. Results PCR and sequencing confirmed that CDC25B3 shRNA lentiviral vector LV-shCDC25B3 was successfully constructed. The titer of the concentrated virus suspension was 1 × 10 8 TU / ml. Conclusion The human CDC25B3 RNAi lentiviral vector was successfully constructed.