[1]方针强,叶钢,欧阳一辛,等.人BMP-7基因重组腺病毒的构建与鉴定[J].第三军医大学学报,2008,30(02):153-156.
 FANG Zhen-qiang,YE Gang,OUYANG Yi-xin,et al.Construction and identification of human BMP-7 gene recombinant adenovirus[J].J Third Mil Med Univ,2008,30(02):153-156.
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人BMP-7基因重组腺病毒的构建与鉴定(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
30卷
期数:
2008年第02期
页码:
153-156
栏目:
论著
出版日期:
2008-01-30

文章信息/Info

Title:
Construction and identification of human BMP-7 gene recombinant adenovirus
作者:
方针强叶钢欧阳一辛刘永亮贾维胜冯嘉瑜
第三军医大学新桥医院:泌尿外科,全军肾脏病中心,检验科
Author(s):
FANG Zhen-qiang YE Gang OUYANG Yi-xin LIU Yong-liang JIA Wei-sheng FENG Jia-yu
Renal Diseases Research Center, Department of Urinary Surgery, Department of Laboratory Medicine, Xinqiao Hospital, Third Military Medical University
关键词:
腺病毒骨形成蛋白-7同源重组
Keywords:
adenovirus BMP-7 gene homogenous recombination
分类号:
R373;R394-33
文献标志码:
A
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1.25×1010 PFU/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。
Abstract:
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 1.25×1010 PFU/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.

参考文献/References:

方针强,叶钢,欧阳一辛,等. 人BMP-7基因重组腺病毒的构建与鉴定[J].第三军医大学学报,2008,30(2):153-156.

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更新日期/Last Update: 2008-04-30