CDC25B_3基因RNAi慢病毒载体的构建与鉴定  

Construction and identification of lentiviral vector of RNA interference of CDC25B3 gene

作　　者：张齐[1] 范健 石欣[1] 孔波[1] 肖志[1] 杨正平[1] 

机构地区：[1]东南大学附属中大医院普通外科,南京210009 [2]扬州大学校长办公室

出　　处：《江苏医药》2009年第7期796-799,共4页Jiangsu Medical Journal

基　　金：国家自然科学基金(30500491)

摘　　要：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 a lentiviral vector of RNA interference （RNAi） of CDC25B3 gene. Methods The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein（GFP）. The resulting lentiviral vector containing CDC25B3 shRNA was named LV-sh CDC25Ba, which was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector LV-shCDC25B3, pHelper 1.0 and pHelper 2. 0. All virus stocks were produced by Lipofectamine 2000-mediated transfection. The titer of virus was tested according to the expression level of GFP. Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of CDC25B3 （LV-shCIX225B3） producing CDC25B3 shRNA was constructed successfully. The titer of concentrated virus was 1 × 10^8 TU/ml. Conclusion The lentivirus RNAi vector of CDC25B3 has been constructed successfully.

关 键 词：RNA干扰 CDC25B 胰腺癌 慢病毒 

分 类 号：R394[医药卫生—医学遗传学]