生物工程学报 25 July 2009, 25(7):1035～1041
腺病毒介导IL-24-E1A双基因载体的构建及其体外抑制SMMC-7721肝癌细胞生长作用初探
汪小华1, 缪竞诚2, 谢宇锋2, 盛伟华2, 单云波2, 杨吉成
1 苏州大学附属第一医院, 苏州215001
2 苏州大学医学部基础医学与生物科学学院细胞与分子生物学教研室, 苏州215123
摘 要: 构建IL-24和E1A双基因腺病毒载体, 获得Ad-IL-24-E1A重组腺病毒子, 分析其体外抑瘤作用。PCR及BglⅡ和SalⅠ酶切获得IL-24, 与空载体构建成pAdTrack-IL-24-IRES。Xho I和EcoR V酶切获得E1A片段, 与pAdTrack-IL-24-IRES连接后成功构建pAdTrack-IL-24-IRES-E1A, 同源重组、包装和扩增获得Ad-IL-24-E1A重组病毒子。用50 MOI重组腺病毒感染SMMC-7721肝癌细胞, MTT法测定Ad-IL-24-E1A的细胞生长抑制作用; 流式细胞仪分析细胞凋亡情况。本研究成功获得Ad-IL-24-E1A重组病毒子。与其他组相比, Ad-IL-24-E1A明显抑制肿瘤细胞生长, 感染48 h后凋亡率达52%, 而抑制正常细胞作用不明显。研究提示Ad-IL-24-E1A双基因重组载体明显抑制SMMC-7721肝癌细胞生长。 关键词: 构建, Ad-IL-24-E1A, SMMC-7721肝癌细胞, 抑瘤
Construction of adenovirus vector expressing IL-24 and E1A and its inhibition of SMMC-7721
Xiaohua Wang1, Jingcheng Miao2, Yufeng Xie2, Weihua Sheng2, Yunbo Shan2, and Jicheng Yang
1 No.1 Affiliated Hospital of Soochow University, Suzhou 215001, China
2 Department of Celluar and Molecular Biology, Medical School, Suzhou University, Suzhou 215123, China
Abstract: We constructed the recombinant adenovirus vector expressing IL-24 and E1A (Ad-IL-24-E1A) and investigated the inhibition of Ad-IL-24-E1A on SMMC-7721 hepatocellular carcinoma in vitro. We amplified IL-24 gene by PCR using pAdTrack-IL-24 as template. The IL-24 gene was cloned into pAdTrack-IRES at the Bgl II and Sal I site to form pAdTrack-IL-24-IRES. E1A digested from pAdTrack-E1A was cloned into the pAdTrack-IL-24-IRES at the Xho I and EcoR V site to form the pAdTrack-IL-24-IRES-E1A. We co-transformed both pAdTrack-IL-24-IRES-E1A and pAdeasy-1 digested by Pme I and packaged to obtain Ad-IL-24-E1A. Ad-IL-24-E1A at 50 MOI infected SMMC-7721 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay determined cell proliferation. Flow cytometry detected Cell apoptosis. The apoptotic rate of SMMC-7721 cells was 52% 48 h after infection with Ad-IL-24-E1A. The result showed that the growth of SMMC-7721 cells was significantly inhibited by Ad-IL-24-E1A at the MOI of 50.
Keywords: construction, Ad-IL-24-E1A, SMMC-7721, growth-suppression
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