人高迁移率族蛋白组A1基因RNA干扰慢病毒载体的构建与鉴定  

Construction and identification of a lentiviral vector harboring RNAi sequence targeting the human high mobility group A1 gene

作　　者：金志良[1] 孙新臣[1] 成红艳[1] 魏青[2] 何少忠[1] 

机构地区：[1]东南大学附属中大医院肿瘤科,江苏南京210009 [2]江苏省肿瘤医院放疗科,江苏南京210009

出　　处：《医学研究生学报》2008年第5期468-471,I0002,共5页Journal of Medical Postgraduates

基　　金：南京市医学发展重点项目基金资助(批准号:ZKX0427);江苏省自然科学基金前期预研项目资助(批准号:BK2005203)

摘　　要：目的:构建和鉴定人高迁移率族蛋白组A1(HMGA1)基因RNA干扰慢病毒表达载体。方法:针对已经筛选确定的HMGA1基因RNA干扰有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经HpaⅠ和XhoⅠ酶切后的pGCL-GFP载体(含U6启动子和绿色荧光蛋白)连接产生LV-sh HMGA1慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV-sh HMGA1慢病毒载体、pHelper 1.0和pHelper 2.0等3种质粒共转染包装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LV-shHMGA1的慢病毒载体,病毒滴度达5×107TU/ml。结论:人HMGA1基因RNA干扰慢病毒载体的成功构建为进一步应用RNAi技术研究HMGA1基因的功能打下了基础。Objective： To construct and identify a lentiviral vector harboring RNAi sequence targeting the human high mobility group A1 （HMGA1） gene. Methods ： The effective sequence of siRNA targeting the HMGA1 gene 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 pGCL-GFP vector diced by the restriction enzyme of Hpa I and Xho I , which contained the U6 promoter and green fluorescent protein （GFP）. The resulting lentiviral vector containing HMGA1 shRNA was named LV-sh HMGA1 and confirmed by PCR and DNA sequencing. A total of 293T cells were cotransfected with LV-sh HMGA1, pHelper 1.0 and pHelper 2.0. All the virus stocks were produced by Lipofectamine2000-mediated transfection. The titer of the virus was tested according to the expression level of GFP. Results ： PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting the human HMGA1 gene was successfully inserted into the lentiviral vector. The titer of the recombinant lentiviral vector was 5 - 107 TU/ml. Conclusion ： The successful construction of the lentiviral vector of HMGA1 has prepared the ground for further studies on the functions of the HMGA1 gene with the RNAi technique

关 键 词：RNA干扰 高迁移率族蛋白组A1 慢病毒 

分 类 号：Q782[生物学—分子生物学]