【摘要】 目的利用AdEasy腺病毒载体系统构建人骨形成蛋白2(BMP2)基因重组腺病毒并在293E细胞中扩增制备重组病毒。方法自人骨形成蛋白2真核表达载体pcDNA3-hBMP2中酶切出hBMP2基因,插入pAdtrackCMV中构建成腺病毒穿梭质粒pAdtrackCMV-hBMP2,经酶切线性化后,采用电穿孔转化到事先电转化腺病毒骨架质粒pAdEasy的BJ5183大肠杆菌电感受态细菌中,挑选同源重组质粒,酶切线性化重组质粒并转染293E细胞包装成重组病毒颗粒,荧光显微镜观察绿色荧光表达。重组病毒上清感染293细胞,荧光显微镜观察绿色荧光表达。结果经限制性内切酶检测和GFP表达证实成功地构建了携带hBMP2基因的重组腺病毒载体并制备出高滴度重组病毒。结论成功地构建了携带hBMP2基因的重组腺病毒载体,为进一步研究rBMP2基因治疗奠定了基础。更多还原

【Abstract】 Objective To constuct recombinant adenovirus vector carrying the human bone morphogenetic protein-2 (BMP-2) gene and amplify the adenovirus vector in 293E cells.Methods hBMP-2cDNA was inserted into adenovirus shuttle plasmid-pAdtrackCMV to generate a recombinant plasmid pAdtrackCMV-hBMP2,then the PmeⅠ-linearized plasmid pAdtrackCMV-hBMP2 was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.The DNA contained in the identified recombinant plasmid was digested with PacⅠ and transfected into 293E cells to package the adenovirus,followed by identification of the recombinant adenovirus by means of observation of green fluorescence protein expression under fluorescent microscopu.After amplified in 293E cells,the Adenovirus was transfected into 293 cells,and the green fluorescence protein expression was detected.Results Recombinant adenoviral vector hBMP2 vere was constructed successfully,whicH was confirmed by restriction enzyme digestion and GFP expression.Conclusion The recombinant adenoviral vector carring hBMP2 was successfully constructed,and provided the basis of hBMP2 gene therapy.更多还原