生物工程學(xué)報(bào) 25 July 2009, 25(7):1035～1041

腺病毒介導(dǎo)IL-24-E1A雙基因載體的構(gòu)建及其體外抑制SMMC-7721肝癌細(xì)胞生長作用初探

汪小華1, 繆競誠2, 謝宇鋒2, 盛偉華2, 單云波2, 楊吉成

1 蘇州大學(xué)附屬第一醫(yī)院, 蘇州215001

2 蘇州大學(xué)醫(yī)學(xué)部基礎(chǔ)醫(yī)學(xué)與生物科學(xué)學(xué)院細(xì)胞與分子生物學(xué)教研室, 蘇州215123

摘 要: 構(gòu)建IL-24和E1A雙基因腺病毒載體, 獲得Ad-IL-24-E1A重組腺病毒子, 分析其體外抑瘤作用,。PCR及BglⅡ和SalⅠ酶切獲得IL-24, 與空載體構(gòu)建成pAdTrack-IL-24-IRES,。Xho I和EcoR V酶切獲得E1A片段, 與pAdTrack-IL-24-IRES連接后成功構(gòu)建pAdTrack-IL-24-IRES-E1A, 同源重組,、包裝和擴(kuò)增獲得Ad-IL-24-E1A重組病毒子,。用50 MOI重組腺病毒感染SMMC-7721肝癌細(xì)胞, MTT法測定Ad-IL-24-E1A的細(xì)胞生長抑制作用; 流式細(xì)胞儀分析細(xì)胞凋亡情況,。本研究成功獲得Ad-IL-24-E1A重組病毒子,。與其他組相比, Ad-IL-24-E1A明顯抑制腫瘤細(xì)胞生長, 感染48 h后凋亡率達(dá)52%, 而抑制正常細(xì)胞作用不明顯。研究提示Ad-IL-24-E1A雙基因重組載體明顯抑制SMMC-7721肝癌細(xì)胞生長,。 關(guān)鍵詞: 構(gòu)建, Ad-IL-24-E1A, SMMC-7721肝癌細(xì)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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