摘要
目的构建表达多药耐药基因1（MDR1）的重组腺病毒载体，体外实验研究MDR1基因对骨髓的保护作用。方法用基因工程技术将MDR1的cDNA亚克隆至穿梭质粒pAdTrack-CMV上，利用PAdEasy系统进行细菌内同源重组，然后通过脂质体将正确重组体包裹并转染293细胞，并在其中包装、扩增、收获病毒。进一步采用PCR方法对重组腺病毒进行鉴定，利用穿梭质粒中所带绿色荧光蛋白GFP报告基因，对病毒滴度进行检测。将收集的重组腺病毒Ad5-MDR1感染小鼠单个核细胞，通过荧光显微镜、免疫组化及流式细胞仪对感染效率及保护作用进行检测，并用Westernblot法检测P-gp的表达。结果酶切鉴定及PCR结果证明多药耐药基因重组腺病毒载体构建成功，病毒滴度达8．3×10^11pfu／ml。对小鼠单个核细胞的感染效率可达10％～15％，转染小鼠单个核细胞48h后，有P-gp蛋白的明显表达。结论成功构建了表达MDR1的重组腺病毒载体，证实MDR转染对骨髓具有保护作用。
Objective To investigate the protective effects of the recombinant adenovirus encoding human multidrug resistance gene （MDR1） for the bone marrow. Methods The MDR1 gene fragment was cloned into the shuttle plasmid pAdTrack-CMV to form the transfer vector by the method of homogenous recombination in bacteria. Then the recombinant adenovirus was transfected into 293cells using lipidosome. The target gene was detected by polymerase chain re-action（PCR）. The titers were determined using the green fluorescent protein （GFP） expression in the shuttle plasmid. The mononuclear cell of mouse were infected with AdS-MDR1. The efficiency of infection and protection were determined by fluorescence microscope immunohistochemistry and flow cytometry. P-gp was detected by Western blotting. Results Restriction endonuclease and PCR analysis confirmed that the MDR1 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 8. 3 × 10^11 pfu/ml. The efficiency of infection to mononuclear cell of mouse achieved 10% - 15%. The expression of MDR1 gene was observed at 48 hours after transfection of the mononuclear cell of mouse with adenovirus particles. Conclusions The protective effect of MDR1 gene for the bone marrow can be achieved.
出处
《中华小儿外科杂志》
CSCD
北大核心
2007年第2期59-63,共5页
Chinese Journal of Pediatric Surgery
基金
国家自然科学基金重点项目（30330590）
