肿瘤特异增殖型腺病毒穿梭质粒pAd-delE1b55kD的构建

Construction of replication-competent adenovirus shuttle plasmids pAd-delE1b55kD

目的:构建一种对肿瘤细胞有特异靶向性的高效载体-肿瘤特异增殖型腺病毒的穿梭质粒pAd-delE1b55kD。方法:利用二步法PCR等基因重组技术,将质粒pXC1编码E1B-55kD蛋白的基因区域缺失,并保留一个BglII酶切位点,以便插入外源性基因;利用凝胶图像分析和基因测序验证所构建质粒的正确性。结果:质粒pXC1的基因序列中,编码E1B-55kD蛋白的基因区中的2279～3326bp区域缺失,该区域上游的序列中第2024位碱基C点突变为T,第2252位碱基C点突变为T及第2261位碱基G点突变为T,从而在该区域上游插入了终止密码子TGA、TAG和TAA。在终止密码子下游,保留了一个BglII酶切位点。结论:成功构建了肿瘤特异增殖型腺病毒穿梭质粒载体pAd-delE1b55kD,为肿瘤的基因治疗提供了一种对肿瘤细胞有特异靶向性的高效载体的穿梭质粒。

Objective： To construct a replication-competent adenovims shuttle plasmids named pAd-delElb55kD, which can transfectthe cell of tumor targeted. Methods： delete the EIB55 region of pXCI and retain a site for Bgl II by such technique of gene recombinationas nested PCR; verify the correctness of pAd-delElb55kD by gel image analysis and sequencing the gene order. Resu/ts： The 2279-3326region of EIB in pXCI was deleted. The 2024 basic radical C was mutated to T, the 2252 basic radical C to T and the 2261 basic radical Gto T. Stop codon TGA ,TAG and TAA were inserted. A site for Bgl Ⅱwas retained in downstream of the stop codon. Conclusion： Thereplication-competent adenovims shuttle plasmids named pAd-deLE1b55kD is constructed, which provided a carrier for the gene therapyof tumor targeted.