Ad-ING4-poly（A）-Promoter-IL-24双基因共表达载体构建及表达  

The anti-tumor effect by adenovirus-mediated ING4 and IL-24 co-expression on hepatocellular carcinoma in vitro

作　　者：盛伟华[1] 谢宇锋[1] 缪竞诚[1] 顾范博[1] 单云波[1] 刘铁连[1] 井莹莹[1] 胡志清[1] 杨吉成[1] 

机构地区：[1]苏州大学医学部细胞与分子生物学教研室,215123

出　　处：《中华微生物学和免疫学杂志》2010年第8期695-703,共9页Chinese Journal of Microbiology and Immunology

基　　金：江苏省卫生厅医学科研基金资助项目（H200914）

摘　　要：目的 构建poly（A）-Promoter介导的携带人肿瘤生长抑制因子4（ING4）和人白细胞介素-24（IL-24）双基因的重组腺病毒共表达载体Ad-ING4-poly（A）-Promoter-IL-24（简称Ad-ING4-IL-24）,研究Ad-ING4-IL-24对HepG-2人肝癌细胞生长的影响.方法 以含有poly（A）和Promoter （hEF1-eIF4g）基因片段的pORF-mbcl-2α质粒为模板,PCR扩增poly（A）-Promoter目的 片段（含Sal Ⅰ,Not Ⅰ）,亚克隆至pAdTrack-CMV载体中以构建pAdTrack-CMV-poly（A）-Promoter空载体,再分别以pcDNA3.0-ING4和pcDNA3.0-IL-24重组质粒为模板,PCR扩增ING4（含BglⅡ,Sal Ⅰ）和IL-24（含XhoⅠ,XbaⅠ）目的 基因片段,并依次插入pAdTrack-CMV-poly（A）-Promoter载体中构建pAdTrackCMV-ING4-Poly（A）-Promoter-IL-24双基因共表达重组转移载体,测序正确后用Pme Ⅰ酶线性化,与腺病毒骨架质粒pAdEasy-1构建同源重组腺病毒质粒pAdEasy-1-pAdTrack-CMV-ING4-poly（A）-Promoter-IL-24,再经Pac Ⅰ酶线性化后用脂质体转染QBI-293A包装细胞,经多轮扩增后收获Ad-ING4-IL-24重组腺病毒子,其效价可达3.5×109PFU/ml,用RT-PCR和Western blot法分别鉴定ING,4和IL-24基因在HepG-2人肝癌细胞中的转录和表达,MTT法和流式细胞仪检测Ad-ING4-IL-24对HepG-2人肝癌细胞生长抑制和诱导细胞凋亡的功能及其增效作用.结果 DNA测序结果显示pAdTrack-CMV转移载体中插入的ING4、poly（A）-Promoter和IL-24序列与GenBank报道的完全一致,RT-PCR和Western blot检测结果显示Ad-ING4-IL-24能成功介导ING4和IL-24基因在HepG-2人肝癌细胞中表达 并能明显抑制HepG-2人肝癌细胞的生长和诱导其凋亡,其中Ad-ING4-IL-24双基因组更优于相应各单基因组.结论 成功构建了poly（A）-Promoter介导的Ad-ING4-IL-24双基因共表达重组腺病毒载体,Ad-ING4-IL-24不仅能明显抑制HepG-2人肝癌细胞生长和诱导其凋亡,而且与Ad-ING4和AdIL-24单基因组相比具有抑癌增效作用.Objective To construct a recombinant adenoviral vector carrying and co-expressing human inhibitor of growth 4（ING4） and human interleukin-24（IL-24） mediated by poly（ A）-Promoter[Ad-ING4-poly（A）-Promoter-IL-24, referred to as Ad-ING4-IL-24] and explore its effect on the growth of HepG-2 human hepatocellular carcinoma cellsin vitro. Methods The poly（A）-Promoter（hEFl-elF4g） （Sal Ⅰ and Not Ⅰ ）, ING4 （ Bgl Ⅱ and Sal Ⅰ ）, and IL-24 （ Xho Ⅰ and Xba Ⅰ ） fragments were amplified by PCRusing pORF-mbcl-2α, pcDNA3.0-IL-24, and pcDNA3.0-ING4 plasmids as templates and subcloned into pAdTrack-CMV transfer vector to form pAdTrack-CMV-ING4-poly （A）-Promoter-lL-24, respectively. The pAdTrack-CMV-ING4-poly （A）-Promoter-IL-24 transfer vector linearized with Pme Ⅰ digestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination. The resultant pAdEasy-l-pAdTrack-CMV-ING4-poly （ A ）-Promoter-IL-24 homologous recombinant plasmids were linearized with Pac Ⅰ digestion and transfected into the human embryonic kidney 293 （QBI-293A） cells by liposome, leading to formation of the recombinant adenoviruses Ad-ING4-IL-24co-expressing ING4 and IL-24. The Ad-ING4-IL-24 were amplified in QBI-293A cells and its titer was up to 3.5 × 109 PFU/ml. Adenovirus-mediated ING4 and IL-24 genes expression in HepG-2 cells was examined by RT-PCR and Western blot. The growth-suppressing and apoptosis-inducingg effect of Ad-ING4-IL-24 coexpressing ING4 and IL-24 on HepG-2 human hepatocellular carcinoma cells was assessed by MTT assay and FCM, respectively. Results DNA sequencing showed that the ING4, poly （A）-Promoter, and IL-24 fragments subcloned into pAdTrack-CMV plasmids were completely identical to those reported in GenBank.ING4 and IL-24 gene mediated by adenovirus could both successfully express in HepG-2 cells. Adenovirusmediated ING4 and IL-24 co-expression significantly suppressed HepG-2 hepatocellular carcinom

关 键 词：腺病毒 生长抑制因子4 IL-24 肿瘤基因治疗 

分 类 号：R346[医药卫生—基础医学]