为开展肿瘤的复合基因治疗,构建以串联方式携带人野生型p53和B7-I基因的重组腺病毒穿梭质粒pXC53/B7-I.将pXC53/B7-1与腺病毒包装质粒GT4050共转染293细胞,通过细胞内同源重组获得重组腺病毒Ad-p53/B7-1.在293细胞中扩增病毒,并通过氯化铯密度梯度超速离心纯化病毒,获得高滴度和高纯度的病毒.分别经免疫组织化学分析和流式细胞分析检测Ad-p53/B7-l介导的人野生型p53和B7-I基因在喉癌细胞Hep-2中的表达.结果表明Ad-p53/B7-1能够有效地将其所携带的目的基因导入Hep-2细胞并使其在细胞中高效表达.%In order to develop a combined gene therapy paradigm for the treatment of cancer, a recombinant adenoviral shuttle plasmid pXC53/B7-1 containing human wild-type p53 and B7-1 genes was constructed by molecular cloning.Recombinant adenoviruse Ad-p53/B7-1 was constructed by homologous recombination of pXC53/B7-1 and adenoviral packaging plasmid GT4050 in 293 cells. Adenoviruses with high titer and purity were obtained by amplifying in 293 cells on a large scale and ultra-centrifugating in CsCI step gradient solutions. The expressions of p53 and B7-1 genes in human laryngeal epithelial carcinoma cells Hep-2 were determined respectively by immunohistochemical assay and flow cytometric analysis. The results showed that human wild-type p53 and B7-1 genes could be efficiently expressed in Hep-2 cells mediated by Ad-p53/B7-1.
展开▼
