人BMP-7基因重组腺病毒的构建与鉴定  

Construction and identification of human BMP-7 gene recombinant adenovirus

作　　者：方针强[1] 叶钢[1] 欧阳一辛[2] 刘永亮[1] 贾维胜[1] 冯嘉瑜[1] 

机构地区：[1]第三军医大学新桥医院泌尿外科,全军肾脏病中心,重庆400037 [2]第三军医大学新桥医院检验科,重庆400037

出　　处：《第三军医大学学报》2008年第2期153-156,共4页Journal of Third Military Medical University

摘　　要：目的构建含有人骨形态发生蛋白-7(bone morphogenetic protein-7,BMP-7)基因的重组腺病毒载体,并对其相关指标进行鉴定,观察其对肾小管上皮细胞株(HKC)细胞的转染情况。方法用基因工程技术将人BMP-7基因cDNA亚克隆至穿梭质粒pDC316上,利用脂质体介导的方法将AdMax腺病毒包装系统的骨架质粒pBHGlox_E1,3Cre和穿梭质粒pDC316-BMP-7转染入HEK293细胞,进行同源重组,得到腺病毒重组质粒Ad5-BMP-7,并在其中包装扩增病毒。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,进行病毒滴度的测定和对HKC细胞感染效率的检测。结果酶切鉴定及PCR结果证明BMP-7基因重组腺病毒载体构建成功,病毒滴度达1.25×1010PFU/ml,对HKC有较强感染能力。结论应用细胞内同源重组方法成功构建了含人BMP-7基因的重组腺病毒载体。Objective To construct and identify the recombinant adenovirus encoding human BMP-7 gene and observe its ability to infect HKC cells. Methods The human BMP-7 gene fragment was cloned into the shuttle plasmid pDC316 to form pDC316-BMP-7. Then pDC316-BMP-7 and the framework plasmid pBHGlox_ E1,3Cre were transfected into HEK293 cells for homogenous recombination in cells with Lipofectamine 2000. The target gene was detected by polymerase chain reaction （PCR）. The titer and its infection rate were determined using the green fluorescent protein （GFP） expression in the shuttle plasmid. Results Restriction endonuclease and PCR analysis confirmed that the human BMP-7 gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was 1.25 ×10^10PFU/ml. The adenovirus has a strong effect on human HKC cells. Conclusion The recombinant adenovirus containing human BMP-7 gene was successfully constructed by the method of homogenous recombination in cells.

关 键 词：腺病毒 骨形成蛋白-7 同源重组 

分 类 号：R373[医药卫生—病原生物学] R394-33[医药卫生—基础医学]