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目的构建由人MDR1启动子调控CD∷upp融合基因表达的重组腺病毒,为对耐药肿瘤体内外靶向基因治疗奠定基础。方法自行设计一对含XhoⅠ和HindⅢ酶切位点的MDR1引物,从外周血中提取人类基因组DNA,通过PCR扩增MDR1启动子并插入PGL3-enhancer载体上,获得PGL3-MDR1质粒。从PORF-CD∷upp质粒中切下CD∷upp基因,插入PGL3-MDR1中MDR1启动子下游,并从中切下目的基因MDR1-CD∷upp,克隆到腺病毒穿梭质粒中,将带目的基因的穿梭质粒pAdTrack-MDR1-CD∷upp。与含有pAdeasy-1的BJ5183菌电转化,筛选提取>30 kb的阳性克隆,经线性化后转染293细胞,通过观察绿色荧光蛋白(GFP)的表达及PCR扩增出重组腺病毒中的目的基因MDR1、CD∷upp等方法加以鉴定。结果成功构建了含MDR1-CD∷upp靶向自杀基因的重组腺病毒载体Ad-MDR1-CD∷upp,病毒滴度为3.0×1010pfu/ml。结论该重组腺病毒的构建为下一步研究其对MDR1耐药细胞系的特异性杀伤作用和对耐药肿瘤模型的靶向基因治疗提供基础。
Objective To construct a recombinant adenovirus that regulates the expression of CD :: upp fusion gene by human MDR1 promoter, which lays a foundation for targeted gene therapy in drug-resistant tumors both in vitro and in vivo. Methods A pair of MDR1 primers containing Xho Ⅰ and Hind Ⅲ restriction sites were designed. Human genomic DNA was extracted from peripheral blood. The MDR1 promoter was amplified by PCR and inserted into PGL3-enhancer vector to obtain PGL3-MDR1 plasmid. The CD :: upp gene was cut from the PORF-CD :: upp plasmid, inserted into the downstream of the MDR1 promoter in PGL3-MDR1, and the target gene MDR1-CD :: upp was excised therefrom and cloned into the adenovirus shuttle plasmid. The target gene Shuttle plasmid pAdTrack-MDR1-CD :: upp. And BJ5183 containing pAdeasy-1. The positive clones were screened and extracted> 30 kb. The positive clones were selected and transfected into 293 cells. The expression of green fluorescent protein (GFP) and PCR amplification of the recombinant adenovirus Gene MDR1, CD :: upp and other methods to be identified. Results The recombinant adenovirus Ad-MDR1-CD ::upp containing MDR1-CD :: upp targeted suicide gene was successfully constructed and the virus titer was 3.0 × 1010pfu / ml. Conclusion The construction of this recombinant adenovirus will provide the foundation for further study of its specific killing effect on MDR1-resistant cell line and targeted gene therapy of drug-resistant tumor model.