新型表达骨形态发生蛋白2腺病毒真核细胞载体的构建和鉴定  

Construction and Identification of a Novel Expressing BMP2 Recombinant Adenovirus Vector

作　　者：张正[1] 刘丹平[1] 陈峻江[1] 郭韬[1] 张男[1] 

机构地区：[1]辽宁医学院附属第一医院骨科,辽宁锦州121000

出　　处：《辽宁医学院学报》2009年第5期392-395,399,477,共6页Journal of Liaoning Medical University (LNMU) Bimonthly

摘　　要：目的构建同时表达具有抗原表位FLAG标记的骨形态发生蛋白2(BMP2)目的蛋白和报告分子绿色荧光蛋白(GFP)的新型腺病毒真核细胞表达栽体。方法将去除翻译终止密码子并添加新的酶切识别位点Xho I的BMP2基因定向连入腺病毒穿梭质粒pShuttle CMV-IRES-hrGFP-1。携带BMP2^+基因的重组腺病毒穿梭质粒经酶切鉴定、Pme I酶切线性化后转化BJ5183-AD-1大肠杆菌,利用细菌内同源重组机制将BMP2+和GFP基因连同其顺势表达元件重组入腺病毒基因组质粒。用Pac I酶切去除其质粒成分,暴露腺病毒基因组的反向末端重复序列,转染293细胞,进行腺病毒的包装,完成新型腺病毒载体Ad CMV-BMP2+-IRES-GFP-1的构建。作为阳性对照,同时制备多年来广为应用的BMP2腺病毒表达载体Ad CMV-BMP2。以两种载体感染骨髓基质细胞,通过RT-PCR和荧光显微镜分别检测骨形态发生蛋白2和绿色荧光蛋白表达情况;通过碱性磷酸酶活性检测判断BMP2诱导成骨分化作用。结果突变后骨形态发生蛋白2基因序列、重组腺病毒穿梭质粒和重组腺病毒质粒酶切图谱符合预定设计。感染骨髓基质细胞后,新型腺病毒载体可同时表达报告分子GFP和具有诱导成骨分化活性的BMP2。结论成功构建表达BMP2的新型腺病毒载体。Objective To construct a novel recombinant adenovirus vector expressing the BMP2 fused to FLAG epitope and green fluorescent protein （GFP） as a reporter on the same transcript. Methods The basic pairs of BMP2 behind the translation stopping codon TAG were removed and a Xho I restriction site was added following the end of the mutant. Then, the mutant of BMP2 gene （ BMP2 ＋ gene） was ligated into the multiple cloning sites of the adenovirus shuttle plasmid pShuttle CMV - IRES - hrGFP - 1 by means of the directional cloning technology. Once tested by restriction digestion, the shuttle plasmid was linearized with Pme I and transformed into BJ5183 - AD - 1 cells to recombine the genes of BMP2 ＋ and GFP together with their cis - acting elements into adeno- viral plasmid through a new simplified bacterial homologous recombinant method. To expose its inversted terminal repeats, the recombinant adenoviral plasmids were digested with Pac I , and were then used to 293 transfected cells to generate the desired recombinant adenoviruses, Ad CMV - BMP2 ＋ - IRES - GFP - 1. After the resultant viruses infected the bone marrow stromal cells （ MSCs）, the expression of BMP - 2 gene was verified by RT - PCR, the osteoinduction activity of the expressed BMP2 was confirmed by alkaline phosphatase activity assay, and the expression of GFP gene was tested through fluorescence microscope. As a positive contrast, another recombinant adenovirus, AdCMV -BMP2, which has been used extensively since many years ago, was also constructed. Results Either the sequence of BMP2 gene mutant or the restriction maps of the recombinant adenoviral shuttle plasmids and the recombinant adenoviral plasmids accorded with experimental project beforehand. The new recombinant adenovirus vectors could express both the gene of BMP2 and GFP at the same time. The expressed BMP2 had osteoinduction activity. Conclusions A novel recombinant adenovirus vector expressing the BMP2 fused to FLAG epitope and green fluorescent protein as a reporter on the s

关 键 词：骨形态发生蛋白2基因 绿色荧光蛋白 腺病毒载体 同源重组 成骨诱导 

分 类 号：R687[医药卫生—骨科学]