共表达人p53、GM-CSF和B7-1基因的重组腺病毒的构建

 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、GM CSF和B7 1基因的重组腺病毒穿梭质粒pBB 1 0 2 .将pBB 1 0 2与腺病毒包装质粒GT40 50共转染 2 93细胞 ,通过细胞内同源重组获得重组腺病毒BB 1 0 2 .在 2 93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、ELISA和流式细胞分析 ,检测BB 1 0 2介导的人野生型p53、GM CSF和B7 1基因在喉癌细胞Hep 2中的表达 .结果表明 ,BB 1 0 2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 ,表达高峰期为转染后 2～ 4d ,此后随时间递减 ,可持续 1 0d以上 .

Construction of Recombinant Adenovirus Co-expressing Human Wild-type p53,GM-CSF and B7-1 Genes

In order to develop a combined gene therapy paradigm for the treatment of cancer,a recombinant adenoviral shuttle plasmid pBB\|102 containing human wild\|type p53,GM\|CSF and B7\|1 genes was constructed by molecular cloning.Recombinant adenovirus BB\|102 was constructed by homologous recombination of pBB\|102 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 CsCl step gradient solutions.The expressions of p53,GM\|CSF and B7\|1 genes in human laryngeal epithelial carcinoma cells Hep\|2 were determined respectively by immunohistochemical assay,ELISA and flow cytometric analysis.The results showed that human wild\|type p53,GM\|CSF and B7\|1 genes could be efficiently expressed in Hep\|2 cells mediated by BB\|102.The peak expression period was on the second to fourth day after infection and then tended to decrease.However,on the tenth day a relatively low expression could still be detected.These results provided a basis for further experimental and clinical studies of combined gene therapy for laryngeal and other cancers.