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目的构建人端粒酶逆转录酶(hTERT)启动子介导的E1A和IL-24双基因靶向腺病毒载体,获得Ad-h-E1A-IL-24靶向重组病毒子,并探索其体外抑瘤作用。方法运用PCR扩增MCF-7乳腺癌细胞的DNA,XbaⅠ和HindⅢ酶切获得280 bp hTERT启动子,插入空载体构建成pTrack-hTERT;运用PCR及HindⅢ和XhoⅠ酶切获得E1A,与pTrack-hTERT构成pAdTrack-hTERT-E1A;运用PCR及NotⅠ和SalⅠ酶切获得IL-24,插入pTrack-PP-IRES;XbaⅠ和XhoⅠ酶切获得hTERT-E1A,与pTrack-PP-IL-24-IRES形成pTrack-hTERT-E1A-PP-IL-24-IRES,同源重组、包装和扩增获得Ad-h-E1A-IL-24重组靶向病毒子。用25 MOI重组靶向腺病毒感染SMMC-7721肝癌细胞,MTT法测定Ad-h-E1A-IL-24的细胞生长抑制作用。结果成功构建pTrack-hTERT-E1A-PP-IL-24-IRES,并获得Ad-h-E1A-IL-24重组病毒子。与非靶向双基因比较,Ad-h-E1A-IL-24组可明显抑制肿瘤细胞生长(P<0.05或0.01)。结论Ad-h-E1A-IL-24靶向腺病毒载体的体外抑瘤作用优于非靶向双基因载体。
Objective To construct double-targeting adenoviral vector mediated by human telomerase reverse transcriptase (hTERT) promoter and obtain Ad-h-E1A-IL-24 targeted recombinant virus and explore its in vitro Anti-tumor effect. Methods The DNA of MCF-7 breast cancer cells was amplified by PCR. The 280 bp hTERT promoter was digested with Xba I and Hind III and inserted into empty vector to construct pTrack-hTERT. E1A was digested with Hind Ⅲ and Xho Ⅰ, IL-24 was inserted into pTrack-PP-IRES by digestion with PCR and NotⅠand SalⅠ, hTERT-E1A was digested with XbaⅠand XhoⅠ, and pTrack-PP-IL- hTERT-E1A-PP-IL-24-IRES, homologous recombination, packaging and amplification of Ad-h-E1A-IL-24 recombinant targeting virus. SMMC-7721 hepatoma cells were infected with recombinant adenovirus at 25 MOI, and the cell growth inhibition of Ad-h-E1A-IL-24 was determined by MTT assay. Results The pTrack-hTERT-E1A-PP-IL-24-IRES was successfully constructed and the Ad-h-E1A-IL-24 recombinant virus was obtained. Ad-h-E1A-IL-24 group significantly inhibited tumor cell growth compared with non-target double gene (P <0.05 or 0.01). Conclusion Ad-h-E1A-IL-24 targeting adenovirus vector in vitro anti-tumor effect than non-target double gene vector.